Immunogenic glycoprotein conjugates

ABSTRACT

The present invention relates generally to glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a spacer containing ((2-oxoethyl)thio)). In an aspect the invention provides oxo-eT linked glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a ((2-oxoethyl)thio) spacer having the formula (I): (I) wherein: A is a group (C═X) m  wherein X is S or O and m is 0 or 1; B is a bond, O, or CH 2 ; and when m is 0, B can also be (C═O); R is a C 2 -C 16  alkylene, C 2 -C 16  heteroakylene, NH—C(═O)—C 2 -C 16  alkylene, or NH—C(═O)—C 2 -C 16  heteroakylene, wherein said alkylene and heteroalkylene are optionally substituted by 1, 2 or 3 groups independently selected from COOR′ where R′ is selected from H, methyl, ethyl or propyl. The invention further relates to immunogenic compositions comprising such glycoconjugates, and to methods for the preparation and use of such glycoconjugates and immunogenic compositions.

FIELD OF THE INVENTION

The invention relates generally to glycoconjugates comprising asaccharide covalently conjugated to a carrier protein through a spacercontaining ((2-oxoethyl)thio) (“oxo-eT” thereafter) having the generalformula (I)

wherein B, A and R have the meaning indicated below, to immunogeniccompositions comprising such glycoconjugates, and to methods for thepreparation and use of such glycoconjugates and immunogeniccompositions.

In an embodiment, said spacer is not the(2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

BACKGROUND OF THE INVENTION

The approach to increasing immunogenicity of poorly immunogenicmolecules by conjugating these molecules to “carrier” molecules has beenutilized successfully for decades (see, e.g., Goebel et al. (1939) J.Exp. Med. 69: 53). For example, many immunogenic compositions have beendescribed in which purified capsular polymers have been conjugated tocarrier proteins to create more effective immunogenic compositions byexploiting this “carrier effect.” Schneerson et al. (1984) Infect.Immun. 45: 582-591). Conjugation has also been shown to bypass the poorantibody response usually observed in infants when immunized with a freepolysaccharide (Anderson et al. (1985) J. Pediatr. 107: 346; Insel etal. (1986) J. Exp. Med. 158: 294).

Conjugates have been successfully generated using various cross-linkingor coupling reagents, such as homobifunctional, heterobifunctional, orzero-length crosslinkers. Many methods are currently available forcoupling immunogenic molecules, such as saccharides, proteins, andpeptides, to peptide or protein carriers. Most methods create amine,amide, urethane, isothiourea, or disulfide bonds, or in some casesthioethers. A disadvantage to the use of cross-linking or couplingreagents which introduce reactive sites into the side chains of reactiveamino acid molecules on carrier and/or immunogenic molecules is that thereactive sites, if not neutralized, are free to react with any unwantedmolecule either in vitro (thus potentially adversely affecting thefunctionality or stability of the conjugates) or in vivo (thus posing apotential risk of adverse events in persons or animals immunized withthe preparations). Such excess reactive sites can be reacted or“capped”, so as to inactivate these sites, utilizing various knownchemical reactions, but these reactions may be otherwise disruptive tothe functionality of the conjugates. This may be particularlyproblematic when attempting to create a conjugate by introducing thereactive sites into the carrier molecule, as its larger size and morecomplex structure (relative to the immunogenic molecule) may render itmore vulnerable to the disruptive effects of chemical treatment. Thus,there remains a need for new methods to prepare appropriately cappedcarrier protein conjugates, such that the functionality of the carrieris preserved and the conjugate retains the ability to elicit the desiredimmune response.

SUMMARY OF THE INVENTION

The present invention relates generally to glycoconjugates comprising asaccharide covalently conjugated to a carrier protein through a spacercontaining ((2-oxoethyl)thio)) (also named “oxo-eT” thereafter).

In an aspect, the present invention relates to glycoconjugatescomprising a saccharide covalently conjugated to a carrier proteinthrough a spacer containing ((2-oxoethyl)thio) having the generalformula (I):

wherein:A is a group (C═X)_(m) wherein X is S or O and m is 0 or 1;B is a bond, O, or CH₂; and when m is 0, B can also be (C═O);R is a C₂-C₁₆ alkylene, C₂-C₁₆ heteroakylene, NH—C(═O)—C₂-C₁₆ alkylene,or NH—C(═O)—C₂-C₁₆ heteroakylene, wherein said alkylene andheteroalkylene are optionally substituted by 1, 2 or 3 groupsindependently selected from COOR′ where R′ is selected from H, methyl,ethyl or propyl.

In the above general formula (I), C₂-C₁₆ alkylene denotes astraight-chain containing 2 to 16 carbon atoms. Examples of suitable(C₂-C₁₆) alkylene radicals are (CH₂)₂, (CH₂)₃, (CH₂)₄, (CH₂)₅, (CH₂)₆,(CH₂)₇, (CH₂)₈, (CH₂)₉ or (CH₂)₁₀.

In the above general formula (I), heteroalkylene refers to an alkylenegroup as defined herein in which one or more of the carbon atoms is eachindependently replaced with the same or different heteroatom selectedfrom O, S or N. In a preferred embodiment, heteroalkylene refers to analkylene group as defined herein in which one or more of the carbonatoms is each replaced with an oxygen atom. In a preferred embodiment,heteroalkylene refers to an alkylene group as defined herein in which 1,2, 3 or 4 of the carbon atoms is each replaced with an oxygen atom.Example of suitable C₂-C₁₆ heteroalkylene are O—CH₂, O—CH₂—CH₂,CH₂—CH₂—O—CH₂—CH₂, O—CH₂—CH₂—O—CH₂—CH₂, O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂,O—CH₂—CH₂—(N—CH₃)—CH₂—CH₂—O—CH₂—CH₂, CH₂—CH₂—(N—CH₃)—CH₂—CH₂,CH₂—CH₂—S—CH₂—CH₂ . . . .

In a preferred embodiment,

-   -   B is O and A is C(═O), or,    -   B is CH₂ and m is 0, or,    -   B is C(═O) and m is 0, and/or    -   R is a C₂-C₁₀ alkylene, C₂-C₁₀ heteroakylene, NH—C(═O)—C₂-C₁₀        alkylene, or NH—C(═O)—C₂-C₁₀ heteroakylene, wherein said        alkylene and heteroalkylene are optionally substituted by 1, 2        or 3 groups independently selected from COOR′ where R′ is        selected from H, methyl, ethyl or propyl, or,    -   R is a C₂-C₁₀ alkylene, C₂-C₁₀ heteroakylene, NH—C(═O)—C₂-C₁₀        alkylene, or NH—C(═O)—C₂-C₁₀ heteroakylene, wherein said        alkylene and heteroalkylene are optionally substituted by COOH.

In other preferred embodiments,

-   -   B is O and A is C(═O), or,    -   B is CH2 and m is 0, or,    -   B is C(═O) and m is 0, and/or    -   R is selected from the groups consisting of (CH₂)₂, (CH₂)₃,        (CH₂)₄, (CH₂)₅, (CH₂)₆, (CH₂)₇, (CH₂)₈, (CH₂)₉ or (CH₂)₁₉.

In other preferred embodiments,

-   -   B is O and A is C(═O), or,    -   B is CH2 and m is 0, or,    -   B is C(═O) and m is 0, and/or    -   R is selected from the groups consisting of O—CH₂, O—CH₂—CH₂,        CH₂—CH₂—O—CH₂—CH₂, O—CH₂—CH₂—O—CH₂—CH₂,        CO—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂,        CO—CH₂—CH₂—(N—CH₃)—CH₂—CH₂—O—CH₂—CH₂, CH₂—CH₂—(N—CH₃)—CH₂—CH₂        and CH₂—CH₂—S—CH₂—CH₂.

In an embodiment, the glycoconjugates of the present invention comprisea saccharide covalently conjugated to a carrier protein through a spacercontaining ((2-oxoethyl)thio) (also named or “oxo-eT” thereafter) withthe provisio that said spacer is not the(2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer (i.e.,—C(O)NH(CH₂)₂SCH₂C(O)—).

The invention further relates to immunogenic compositions comprisingsuch glycoconjugates, and to methods for the preparation and use of suchglycoconjugates and immunogenic compositions.

In an aspect, the present invention is directed towards methods ofmaking glycoconjugates comprising a saccharide covalently conjugated toa carrier protein through a bivalent, heterobifunctional linker referredto herein as a ((2-oxoethyl)thio) or “oxo-eT” spacer.

In some embodiments, the oxo-eT linked glycoconjugates of the inventionhave the following formulas

-   -   1. a (((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC) linked        glycoconjugate having formula (II)

wherein R is (CH₂)_(n) where n is 3 to 10;

-   -   2. a (((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC) linked        glycoconjugate, having the following formula (III):

wherein R is selected from (CH₂CH₂O)_(m)CH₂CH₂, CH(COOH)(CH₂),NHCO(CH₂)_(n), NHCO(CH₂CH₂O)_(m)CH₂CH₂, OCH₂(CH₂)_(n) orO(CH₂CH₂O)_(m)CH₂CH₂;wherein n is selected from 1 to 10 and m is selected from 1 to 4.

-   -   3. a (((2-oxoethyl)thio)alkyl)amine (oxo-eTAAN) linked        glycoconjugate having formula (IV):

wherein R is selected from CH₂(CH₂)_(n), (CH₂CH₂O)_(m)CH₂CH₂,CH(COOH)(CH₂)_(n), NHCO(CH₂)_(n), NHCO(CH₂CH₂O)_(m)CH₂CH₂, OCH₂(CH₂)_(n)or O(CH₂CH₂O)_(m)CH₂CH₂;wherein n is selected from 1 to 10 and m is selected from 1 to 4;

-   -   4. a (((2-oxoethyl)thio)alkyl)amide (oxo-eTAAD) linked        glycoconjugate having formula (V)

wherein R is selected from CH₂(CH₂)₁, (CH₂CH₂O)_(m)CH₂CH₂,CH(COOH)(CH₂)₁, NHCO(CH₂)₁, NHCO(CH₂CH₂O)_(m)CH₂CH₂, OCH₂(CH₂)_(n) orO(CH₂CH₂O)_(m)CH₂CH₂;wherein n is selected from 1 to 10 and m is selected from 1 to 4.

In a preferred embodiment, the invention relates to a glycoconjugates offormula (I), (II), (III) (IV) or (V) where R is selected from

-   -   (CH2)_(n) wherein n is selected from 3 to 10;    -   (CH₂CH₂O)_(m)CH₂CH₂ wherein m is selected from 1 to 3;    -   CH(COOH)(CH₂)_(n) wherein n is selected from 1 to 8;    -   NHCO(CH₂)_(n) wherein n is selected from 1 to 8;    -   NHCO(CH₂CH₂O)_(m)CH₂CH₂ wherein m is 1 or 2,    -   OCH₂(CH₂)_(n) wherein n is selected from 1 to 8, or,    -   —O(CH2CH₂O)_(m)CH₂CH₂ wherein m is 1 or 2.

In the above defined glycoconjugates comprising a spacer containing((2-oxoethyl)thio) as part of the structural linkage between asaccharide and a carrier protein or peptide, said spacer provides stablethioether and amide bonds.

The glycoconjugates of the present invention comprise a spacercontaining ((2-oxoethyl)thio) as part of the structural linkage betweena saccharide and a carrier protein or peptide, said spacer providesstable thioether and amide bonds. Additionally, it is preferred thatsaid spacer is relatively short so as to lower the risk of generating animmune response against the spacer part of the conjugate. An immuneresponse against the spacer part of the conjugate is not desirable and ashort spacer has the benefit of minimizing said risk. Therefore in anembodiment, the glycoconjugates of the present invention comprise aspacer containing ((2-oxoethyl)thio) where the number of atoms of thatspacer (e.g. contained in the central box of formulas I, II, III, IV orV) is 25 atoms or less. Preferably, the number of atoms of that spacer(e.g. contained in the central box of formulas I, II, III, IV or V) is21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 atoms.Even more preferably, the number of atoms of that spacer (e.g. containedin the central box of formulas I, II, III, IV or V) is 15 or less (suchas 14, 13, 12, 11, 10, 9, 8, 7, or 6 atoms).

The invention further provides oxo-eT linked glycoconjugates,immunogenic compositions comprising them, and methods for the use ofsuch glycoconjugates and immunogenic compositions.

In one aspect, the invention provides a glycoconjugate comprising asaccharide conjugated to a carrier protein through an oxo-eTAC spacer,wherein the saccharide is covalently linked to the oxo-eTAC spacerthrough a carbamate linkage, and wherein the carrier protein iscovalently linked to the oxo-eTAC spacer through a thioether and amidelinkage. In an embodiment, said oxo-eTAC spacer is not the(2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer (i.e.,—C(O)NH(CH2)2SCH2C(O)—).

In one aspect, for the generation of oxo-eTAC spacer in the conjugate,the preferred linkers used herein are Mercaptopropionylhydrazide (MPH,n=2), L-Cystine dimethylester dihydrochloride (n=1), and2-(2-aminoethoxy)ethane-1-thiol (AEET, n=2) and 4-Amino-1-butanethiolhydrochloride (n=4).

In another aspect, the invention provides a glycoconjugate comprising asaccharide conjugated to a carrier protein through an oxo-eTAAN spacer,wherein the saccharide is covalently linked to the oxo-eTAAN spacerthrough an amine linkage, and wherein the carrier protein is covalentlylinked to the oxo-eTAAN spacer through a thioether and amide linkage.

In another aspect, the invention provides a glycoconjugate comprising asaccharide conjugated to a carrier protein through an oxo-eTAAD spacer,wherein the saccharide is covalently linked to the oxo-eTAAD spacerthrough an amide linkage, and wherein the carrier protein is covalentlylinked to the oxo-eTAAD spacer through a thioether and amide linkage.

In some embodiments, the saccharide is a polysaccharide, such as acapsular polysaccharide derived from bacteria, in particular frompathogenic bacteria. In other embodiments, the saccharide is anoligosaccharide or a monosaccharide.

The carrier proteins incorporated into the glycoconjugates of theinvention are selected from the group of carrier proteins generallysuitable for such purposes, as further described herein or known tothose of skill in the art. In particular embodiments, the carrierprotein is CRM₁₉₇.

In another aspect, the invention provides a method of making aglycoconjugate comprising a saccharide conjugated to a carrier proteinthrough an oxo-eTAC spacer, comprising the steps of: a) reacting asaccharide with a carbonic acid derivative or cyanogen derivative toproduce an activated saccharide; b) reacting the activated saccharidewith a bifunctional linker containing amine and thiol functionalities(as protected or free thiol forms, e.g. DL-cystine or DL-cysteine or asalt thereof, or mercaptopropionylhydrazide, or2-(2-aminoethoxy)ethane-1-thiol, or 4-Amino-1-butanethiol or a saltthereof), to produce a thiolated saccharide; c) reacting the thiolatedsaccharide with a deprotecting or reducing agent (if thiol is protected)to produce an activated thiolated saccharide comprising one or more freesulfhydryl residues; d) reacting the activated thiolated saccharide withan activated carrier protein comprising one or more α-haloacetamidegroups, to produce a thiolated saccharide-carrier protein conjugate; ande) reacting the thiolated saccharide-carrier protein conjugate with (i)a first capping reagent capable of capping unconjugated α-haloacetamidegroups of the activated carrier protein; and/or (ii) a second cappingreagent capable of capping unconjugated free sulfhydryl residues of theactivated thiolated saccharide; whereby an oxo-eTAC linkedglycoconjugate is produced.

In frequent embodiments, step a) is done in an organic solvent.

In frequent embodiments, the carbonic acid derivative is1,1′-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1′-carbonyldiimidazole(CDI) or disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidylchloroformate. Preferably, the carbonic acid derivative is CDT and theorganic solvent is a polar aprotic solvent, such as dimethylsulfoxide(DMSO). In preferred embodiments, the thiolated saccharide is producedby reaction of the activated saccharide with a heterobifunctionalthioalkylamine reagent or a salt thereof. The oxo-eTAC linkedglycoconjugates produced by the methods of the invention may berepresented by general formulas (II and III).

In frequent embodiments, the first capping reagent isN-acetyl-L-cysteine, which reacts with unconjugated α-haloacetamidegroups on lysine residues of the carrier protein to form anS-carboxymethylcysteine (CMC) residue covalently linked to the activatedlysine residue through a thioether linkage. In other embodiments, thesecond capping reagent is iodoacetamide (IAA), which reacts withunconjugated free sulfhydryl groups of the activated thiolatedsaccharide to provide a capped thioacetamide. Frequently, step e)comprises capping with both a first capping reagent and a second cappingreagent. In certain embodiments, step e) comprises capping withN-acetyl-L-cysteine as the first capping reagent and IAA as the secondcapping reagent.

In some embodiments, the capping step e) further comprises reaction witha reducing agent, for example, DTT, TCEP, or mercaptoethanol, afterreaction with the first and/or second capping reagent.

In some embodiments, step d) further comprises providing an activatedcarrier protein comprising one or more α-haloacetamide groups prior toreacting the activated thiolated saccharide with the activated carrierprotein. In frequent embodiments, the activated carrier proteincomprises one or more α-bromoacetamide groups.

In another aspect, the invention provides a method of making aglycoconjugate comprising a saccharide conjugated to a carrier proteinthrough an oxo-eTAAN spacer, comprising the steps of: a) reacting asaccharide with an oxidizing reagent to generate aldehyde groups toproduce an activated saccharide; b) reacting the activated saccharidewith a bifunctional linker containing amine and thiol functionalities(in protected or free forms) from the amino end of the linker, toproduce a thiolated saccharide by reductive amination; c) reacting thethiolated saccharide with a deprotecting agent or reducing agent (ifthiol is protected) to produce an activated thiolated saccharidecomprising one or more free sulfhydryl residues; d) reacting theactivated thiolated saccharide with an activated carrier proteincomprising one or more α-haloacetamide groups, to produce a thiolatedsaccharide-carrier protein conjugate; and e) reacting the thiolatedsaccharide-carrier protein conjugate with (i) a first capping reagentcapable of capping unconjugated α-haloacetamide groups of the activatedcarrier protein; and/or (ii) a second capping reagent capable of cappingunconjugated free sulfhydryl residues of the activated thiolatedsaccharide; whereby an oxo-eTAAN linked glycoconjugate is produced. Theoxo-eTAAN linked glycoconjugates produced by the methods of theinvention may be represented by general formula (IV).

In a preferred embodiment of the above for the generation of thethiolated polysaccharide, the primary hydroxyl groups of thepolysaccharide are oxidized by 2,2,6,6-Tetramethyl-1-piperidinyloxy(TEMPO)/N-Chlorosuccinimide (NCS) reagent system (see FIG. 2 orapplication number PCT/IB2013/060933 which is incorporated herein byreference as if set forth in its entirety), prior to further reactionwith the amino end of eTAAN linker.

In another aspect, the invention provides a method of making aglycoconjugate comprising a carboxyl containing saccharide conjugated toa carrier protein through an oxo-eTAAD spacer, comprising the steps of:a) first reacting the carboxyl group containing saccharide to generatedan activated saccharide with a carbodiimide or a derivative thereof; b)reacting the activated saccharide with a heterobifunctional linkercontaining amine and thiol functionalities (in protected or free form)from the amino end, to produce a thiolated saccharide; c) reacting thethiolated saccharide with a deprotecting or reducing agent (ifprotected) to produce an activated thiolated saccharide comprising oneor more free sulfhydryl residues; d) reacting the activated thiolatedsaccharide with an activated carrier protein comprising one or moreα-haloacetamide groups, to produce a thiolated saccharide-carrierprotein conjugate; and e) reacting the thiolated saccharide-carrierprotein conjugate with (i) a first capping reagent capable of cappingunconjugated α-haloacetamide groups of the activated carrier protein;and/or (ii) a second capping reagent capable of capping unconjugatedfree sulfhydryl residues of the activated thiolated saccharide; wherebyan oxo-eTAAD linked glycoconjugate is produced.

In another aspect, the invention provides a method of making aglycoconjugate comprising a carboxyl functionalized saccharideconjugated to a carrier protein through an oxo-eTAAD spacer. As anexample, the primary hydroxyl of the saccharide first could be convertedto carboxyl by reaction with TEMPO/sodium hypochlorite.

In frequent embodiments, the carbodiimide derivative is1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), orN,N′-dicyclohexylcarbodiimide (DCC), or1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide. Preferably, thecarbodiimide derivative is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) and the organic solvent is a polar aprotic solvent, such asdimethylsulfoxide (DMSO). In preferred embodiments, the thiolatedsaccharide is produced by reaction of the activated saccharide with a abifunctional linker containing amine and thiol functionalities Theoxo-eTAAD linked glycoconjugates produced by the methods of theinvention may be represented by general formula (V) In a preferredembodiment of the above for the reaction of the carboxyl containingsaccharide, the initial activation step is accomplished by carbodiimideand thiazolidinone thione, prior to the reaction with the amino end ofthe heterobifunctional eTAAD linker. In another embodiment the initialcarboxylic acid activation step is accomplished byN-ethyl-3-phenylisoxazolium-3′-sulfonate (Woodward's reagent K) prior toreaction with the amino end of the bifunctional eTAAD linker.

In another aspect, the invention provides an oxo-eT linkedglycoconjugate comprising a saccharide conjugated to a carrier proteinthrough an oxo-eT spacer produced according to any of the methodsdisclosed herein.

For each of the aspects of the invention, in particular embodiments ofthe methods and compositions described herein, the oxo-eT linkedglycoconjugate comprises a saccharide which is a bacterial capsularpolysaccharide, in particular a capsular polysaccharide derived frompathogenic bacteria.

By way of example, the polysaccharide may be from Gram negative bacteriaselected from the group consisting of: Escherichia coli, Francisellatularensis, H influenzae, Klebsiella, Moraxella catarrhalis, Neisseriameningitidis, Porphyromonas-A-gingivalis, Pseudomonas aeruginosa,Burkholderia cepacia, Salmonella typhi, Salmonella typhimurium,Salmonella paratyphi, Shigella dysenteriae, Shigella flexneri, Shegellasonnei and Vibrio cholera. The polysaccharide may be from Gram positivebacteria selected from the group consisting of: Enterococcus faecalis,Enterococcus faecium, Group A Streptococcus, Group B Streptococcus,Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcusepidermidis and Streptococcus pneumoniae.

In some embodiments, the oxo-eT linked glycoconjugate comprises apneumococcal (Pn) capsular polysaccharide derived from Streptococcuspneumoniae. In specific embodiments, the Pn capsular polysaccharide isselected from the group consisting of Pn-serotype 1, 2, 3, 4, 5, 6A, 6B,7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22F,23A, 23B, 23F, 33F and 35B capsular polysaccharides. In specificembodiments, the Pn capsular polysaccharide is selected from the groupconsisting of Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F,14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides. Inspecific embodiments, the Pn capsular polysaccharide is selected fromthe group consisting of Pn-serotype 2, 9N, 15A, 17F, 20, 23A, 23B and35B capsular polysaccharides.

In other such embodiments, the oxo-eT linked glycoconjugate comprises ameningococcal (Mn) capsular saccharides (oligo or polysaccharide)derived from Neisseria meningitidis. In specific embodiments, the Mncapsular polysaccharide is selected from the group consisting ofMn-serotype A, C, W135 and Y capsular polysaccharides. In specificembodiments, the Mn capsular polysaccharide is Mn-serotype X capsularpolysaccharide.

In other such embodiments, the oxo-eT linked glycoconjugate comprises acapsular polysaccharide derived from Group B Streptococcus (GBS). Inspecific embodiments, the GBS capsular polysaccharide is selected fromthe group consisting of serotype Ia, Ib, II, III or V capsularpolysaccharides.

In other such embodiments, the oxo-eT linked glycoconjugate comprises acapsular polysaccharide derived from Staphylococcus aureus. In specificembodiments, the S. aureus capsular polysaccharide is S. aureus serotype5 or 8 capsular polysaccharide.

In other such embodiments, the oxo-eT linked glycoconjugate comprises acapsular polysaccharide derived from an Enterococcus bacteria. Inspecific embodiments, the Enterococcus capsular polysaccharide isEnterococcus faecalis or Enterococcus faecium capsular polysaccharide.

In particularly preferred embodiments, the saccharide is a bacterialcapsular polysaccharide, such as a Pn or Mn or GBS or S. aureus orEnterococcus capsular polysaccharide, covalently conjugated to CRM₁₉₇through an oxo-eT spacer.

The compositions and methods described herein are useful in a variety ofapplications. For example, the glycoconjugates of the invention can beused in the production of immunogenic compositions comprising an oxo-eTlinked glycoconjugate. Such immunogenic compositions can be used toprotect recipients from bacterial infections, for example by pathogenicbacteria such as S. pneumonia or N. meningitidis or GBS or S. aureus orEnterococcus.

Thus, in another aspect, the invention provides an immunogeniccomposition comprising an oxo-eT linked glycoconjugate and apharmaceutically acceptable excipient, carrier or diluent, wherein theglycoconjugate comprises a saccharide covalently conjugated to a carrierprotein through an oxo-eT spacer, as described herein.

In frequent embodiments, the immunogenic composition comprises an oxo-eTlinked glycoconjugate and a pharmaceutically acceptable excipient,carrier or diluent, wherein the glycoconjugate comprises a bacterialcapsular polysaccharide.

In some such embodiments, the immunogenic composition comprises anoxo-eT linked glycoconjugate which comprises a Pn capsularpolysaccharide derived from S. pneumoniae. In some specific embodiments,the Pn capsular polysaccharide is selected from the group consisting ofPn-serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15A, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, 33F and 35Bcapsular polysaccharides. In specific embodiments, the Pn capsularpolysaccharide is selected from the group consisting of Pn-serotype 1,3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F,23F and 33F capsular polysaccharides. In specific embodiments, the Pncapsular polysaccharide is selected from the group consisting ofPn-serotype 2, 9N, 15A, 17F, 20, 23A, 23B and 35B capsularpolysaccharides.

In other such embodiments, the immunogenic composition comprises anoxo-eT linked glycoconjugate which comprises a Mn capsularpolysaccharide derived from N. meningitidis. In some specificembodiments, the Mn capsular polysaccharide is selected from the groupconsisting of Mn-serotype A, C, W135 and Y capsular polysaccharides. Inspecific embodiments, the Mn capsular polysaccharide is Mn-serotype Xcapsular polysaccharide.

In other such embodiments, the oxo-eT linked glycoconjugate comprises acapsular polysaccharide derived from Group B Streptococcus (GBS). Inspecific embodiments, the GBS capsular polysaccharide is selected fromthe group consisting of serotype Ia, Ib, II, III or V capsularpolysaccharides.

In other such embodiments, the oxo-eT linked glycoconjugate comprises acapsular polysaccharide derived from Staphylococcus aureus. In specificembodiments, the S. aureus capsular polysaccharide is S. aureus serotype5 or 8 capsular polysaccharide.

In other such embodiments, the oxo-eT linked glycoconjugate comprises acapsular polysaccharide derived from an Enterococcus bacteria. Inspecific embodiments, the Enterococcus capsular polysaccharide isEnterococcus faecalis or Enterococcus faecium capsular polysaccharide.

In preferred embodiments, the immunogenic composition comprises anoxo-eT linked glycoconjugate which comprises a bacterial capsularpolysaccharide, such as a Pn or Mn or GBS or S. aureus or Enterococcuscapsular polysaccharide, covalently conjugated to CRM₁₉₇ through anoxo-eT spacer.

In some embodiments, the immunogenic compositions comprise an adjuvant.In some such embodiments, the adjuvant is an aluminum-based adjuvantselected from the group consisting of aluminum phosphate, aluminumsulfate and aluminum hydroxide. In one embodiment, the immunogeniccompositions described herein comprise the adjuvant aluminum phosphate.

In another aspect, the invention provides a method of preventing,treating or ameliorating a bacterial infection, disease or condition ina subject, comprising administering to the subject an immunologicallyeffective amount of an immunogenic composition of the invention, whereinsaid immunogenic composition comprises an oxo-eT linked glycoconjugatecomprising a bacterial antigen, such as a bacterial capsularpolysaccharide.

In one embodiment, the infection, disease or condition is associatedwith S. pneumonia bacteria and the glycoconjugate comprises a Pncapsular polysaccharide. In another embodiment, the infection, diseaseor condition is associated with N. meningitidis bacteria and theglycoconjugate comprises a Mn capsular polysaccharide. In anotherembodiment, the infection, disease or condition is associated with GroupB Streptococcus bacteria and the glycoconjugate comprises a GBS capsularpolysaccharide. In another embodiment, the infection, disease orcondition is associated with S. aureus bacteria and the glycoconjugatecomprises a S. aureus capsular polysaccharide. In another embodiment,the infection, disease or condition is associated with Enterococcusfaecalis or Enterococcus faecium and the glycoconjugate comprisesrespectively a Enterococcus faecalis or a Enterococcus faecium capsularpolysaccharide.

In other aspects, the invention provides a method for inducing an immuneresponse against pathogenic bacteria; a method for preventing, treatingor ameliorating a disease or condition caused by pathogenic bacteria;and a method for reducing the severity of at least one symptom of aninfection, disease or condition caused by pathogenic bacteria, in eachcase by administering to a subject an immunologically effective amountof an immunogenic composition comprising an oxo-eT linked glycoconjugateand a pharmaceutically acceptable excipient, carrier or diluent, whereinthe glycoconjugate comprises a bacterial antigen, such as a bacterialcapsular polysaccharide derived from the pathogenic bacteria.

In another aspect, the invention provides a method of inducing an immuneresponse in a subject, comprising administering to the subject animmunologically effective amount of an immunogenic compositioncomprising an oxo-eT linked glycoconjugate and a pharmaceuticallyacceptable excipient, carrier or diluent, wherein the glycoconjugatecomprises a bacterial antigen, such as a bacterial capsularpolysaccharide. In preferred embodiments, the method involves producinga protective immune response in the subject, as further describedherein.

In another aspect, the invention provides a method of administering animmunologically effective amount immunogenic composition comprising anoxo-eT linked glycoconjugate to a subject to generate a protectiveimmune response in the subject, as further described herein.

In a further aspect, the invention provides an antibody generated inresponse an oxo-eT linked glycoconjugate of the present invention, or animmunogenic composition comprising such a glycoconjugate. Suchantibodies can be used in research and clinical laboratory assays, suchas bacterial detection and serotyping, or may be used to confer passiveimmunity to a subject.

In yet another aspect, the invention provides an immunogenic compositioncomprising an oxo-eT linked glycoconjugate of the present invention, foruse in the prevention, treatment or amelioration of bacterial infection,for example infection by S. pneumonia or N. meningitidis or Group BStreptococcus or S. aureus or Enterococcus bacteria (such asEnterococcus faecalis or a Enterococcus faecium).

In another aspect, the invention provides the use of an immunogeniccomposition comprising an oxo-eT linked glycoconjugate of the presentinvention, for the preparation of a medicament for the prevention,treatment or amelioration of bacterial infection, for example infectionby S. pneumonia or N. meningitidis or Group B Streptococcus or S. aureusor Enterococcus bacteria (such as Enterococcus faecalis or aEnterococcus faecium).

In certain preferred embodiments of the therapeutic and/or prophylacticmethods and uses described above, the immunogenic composition comprisesan oxo-eT linked glycoconjugate comprising a bacterial capsularpolysaccharide covalently linked to a carrier protein through an oxo-eTspacer. In frequent embodiments of the methods and uses describedherein, the bacterial capsular polysaccharide is a Pn capsularpolysaccharide or a Mn capsular polysaccharide or a GBS capsularpolysaccharide or a S. aureus polysaccharide or an or Enterococcusbacteria polysaccharide (such as Enterococcus faecalis or Enterococcusfaecium). In some such embodiments, the Pn capsular polysaccharide isselected from the group consisting of Pn-serotype 1, 3, 4, 5, 6A, 6B,7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33Fcapsular polysaccharides. In specific embodiments, the Pn capsularpolysaccharide is selected from the group consisting of Pn-serotype 1,2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 17F,18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, 33F and 35B capsularpolysaccharides. In specific embodiments, the Pn capsular polysaccharideis selected from the group consisting of Pn-serotype 1, 3, 4, 5, 6A, 6B,7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33Fcapsular polysaccharides. In specific embodiments, the Pn capsularpolysaccharide is selected from the group consisting of Pn-serotype 2,9N, 15A, 17F, 20, 23A, 23B and 35B capsular polysaccharides. In othersuch embodiments, the Mn capsular polysaccharide is selected from thegroup consisting of Mn-serotype A, C, W135 and Y capsularpolysaccharides. In other such embodiments, the GBS capsularpolysaccharide is selected from the group consisting of serotype Ia, Ib,II, III or V capsular polysaccharides.

In certain embodiments of the therapeutic and/or prophylactic methodsand uses described above, the immunogenic composition comprises anoxo-eT linked glycoconjugate comprising a bacterial capsularpolysaccharide covalently linked to a carrier protein through an oxo-eTspacer. In certain embodiments of the methods and uses described herein,the bacterial capsular polysaccharide is a S. aureus capsularpolysaccharide. In other such embodiments, the S. aureus capsularpolysaccharide is S. aureus serotype 5 or 8 capsular polysaccharide.

In certain embodiments of the therapeutic and/or prophylactic methodsand uses described above, the immunogenic composition comprises anoxo-eT linked glycoconjugate comprising a bacterial capsularpolysaccharide covalently linked to a carrier protein through an oxo-eTspacer. In certain embodiments of the methods and uses described herein,the bacterial capsular polysaccharide is an Enterococcus bacteriacapsular polysaccharide. In other such embodiments, the Enterococcusbacteria capsular polysaccharide is Enterococcus faecalis orEnterococcus faecium capsular polysaccharide.

In certain preferred embodiments, the carrier protein is CRM₁₉₇. Inparticularly preferred embodiments, the immunogenic compositioncomprises an oxo-eT linked glycoconjugate which comprises a bacterialcapsular polysaccharide, such as a Pn or Mn or GBS or S. aureus or anEnterococcus bacteria polysaccharide (such as Enterococcus faecalis orEnterococcus faecium) capsular polysaccharide, covalently conjugated toCRM₁₉₇ through an oxo-eT spacer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a general scheme for the preparation of oxo-eTAC linkedglycoconjugates of the invention, for a glycoconjugate comprising apolysaccharide covalently conjugated to CRM₁₉₇.

FIG. 2 shows a general scheme for the preparation of oxo-eTAAN linkedglycoconjugates of the invention, for a glycoconjugate comprising apolysaccharide covalently conjugated to CRM₁₉₇.

FIG. 3 shows a general scheme for the preparation of oxo-eTAAD linkedglycoconjugates of the invention, for a glycoconjugate comprising apolysaccharide covalently conjugated to CRM₁₉₇.

FIG. 4 shows a general scheme for the preparation of oxo-eTAAD linkedglycoconjugates of the invention, using thiazolidinonethione derivative,for a glycoconjugate comprising a polysaccharide covalently conjugatedto CRM₁₉₇.

FIG. 5 shows a repeating polysaccharide structure of S. pneumoniaeserotype 33F (Pn-33F) capsular polysaccharide.

FIG. 6 shows a repeating polysaccharide structure of S. pneumoniaeserotype 22F (Pn-22F) capsular polysaccharide.

FIG. 7 shows a repeating polysaccharide structure of S. pneumoniaeserotype 10A (Pn-10A) capsular polysaccharide.

FIG. 8 shows a repeating polysaccharide structure of S. pneumoniaeserotype 11A (Pn-11A) capsular polysaccharide.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention pertains. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, certain preferred methods andmaterials are described herein. In describing the embodiments andclaiming the invention, certain terminology will be used in accordancewith the definitions set out below.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless indicated otherwise. Thus, for example, references to“the method” includes one or more methods, and/or steps of the typedescribed herein, and references to “an oxo-eT spacer” refer to one ormore eT spacers, as will be apparent to one of ordinary skill in the artupon reading the disclosure.

As used herein, the term “about” means within a statistically meaningfulrange of a value, such as a stated concentration range, time frame,molecular weight, temperature or pH. Such a range can be within an orderof magnitude, typically within 20%, more typically within 10%, and evenmore typically within 5% of the indicated value or range. Sometimes,such a range can be within the experimental error typical of standardmethods used for the measurement and/or determination of a given valueor range. The allowable variation encompassed by the term “about” willdepend upon the particular system under study, and can be readilyappreciated by one of ordinary skill in the art. Whenever a range isrecited within this application, every whole number integer within therange is also contemplated as an embodiment of the invention.

It is noted that in this disclosure, terms such as “comprises,”“comprised,” “comprising,” “contains,” “containing” and the like canhave the meaning attributed to them in U.S. patent law; e.g., they canmean “includes,” “included,” “including” and the like. Such terms referto the inclusion of a particular ingredients or set of ingredientswithout excluding any other ingredients. Terms such as “consistingessentially of” and “consists essentially of” have the meaningattributed to them in U.S. patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from the novel orbasic characteristics of the invention. The terms “consists of” and“consisting of” have the meaning ascribed to them in U.S. patent law;namely, that these terms are closed ended. Accordingly, these termsrefer to the inclusion of a particular ingredient or set of ingredientsand the exclusion of all other ingredients.

The term “saccharide” as used herein may refer to a polysaccharide, anoligosaccharide, or a monosaccharide. Frequently, references to asaccharide refer to a bacterial capsular polysaccharide, in particularcapsular polysaccharides derived from pathogenic bacteria such as S.pneumoniae or N. meningitis or GBS or S. aureus or an Enterococcusbacteria (such as Enterococcus faecalis or Enterococcus faecium).

The terms “conjugate” or “glycoconjugate” are used interchangeablyherein to refer to a saccharide covalently conjugated to a carrierprotein. The glycoconjugates of the present invention are sometimesreferred to herein as “oxo-eT linked” glycoconjugates, which comprise asaccharide covalently conjugated to a carrier protein through at leastone oxo-eT spacer. The oxo-eT linked glycoconjugates of the inventionand immunogenic compositions comprising them may contain some amount offree saccharide.

The term “free saccharide” as used herein means a saccharide that is notcovalently conjugated to the carrier protein or a saccharide that iscovalently attached to very few carrier proteins attached in a highsaccharide/protein ratio (>5:1), but is nevertheless present in theglycoconjugate composition. The free saccharide may be non-covalentlyassociated with (i.e., non-covalently bound to, adsorbed to, orentrapped in or with) the conjugated saccharide-carrier proteinglycoconjugate. The terms “free polysaccharide” and “free capsularpolysaccharide” may be used herein to convey the same meaning withrespect to glycoconjugates wherein the saccharide is a polysaccharide ora capsular polysaccharide, respectively.

As used herein, “to conjugate,” “conjugated” and “conjugating” refer toa process whereby a saccharide, for example a bacterial capsularpolysaccharide, is covalently attached to a carrier molecule or carrierprotein. In the methods of the present invention, the saccharide iscovalently conjugated to the carrier protein through at least one oxo-eTspacer. The conjugation can be performed according to the methodsdescribed below or by other processes known in the art. Conjugation to acarrier protein enhances the immunogenicity of a bacterial capsularpolysaccharide.

Glycoconjugates

The present invention relates to glycoconjugates comprising a saccharidecovalently conjugated to a carrier protein through one or more oxo-eTspacers, wherein the saccharide is covalently conjugated to the oxo-eTspacer through a carbamate or an amine or an amide linkage, and whereinthe carrier protein is covalently conjugated to the oxo-eT spacerthrough a thioether and an amide linkage.

In addition to the presence of one or more oxo-eT spacers, novelfeatures of the glycoconjugates of the present invention include themolecular weight profiles of the saccharides and resulting oxo-eT linkedglycoconjugates, the ratio of conjugated lysines per carrier protein andthe number of lysines covalently linked to the polysaccharide throughthe oxo-eT spacer(s), the number of covalent linkages between thecarrier protein and the saccharide as a function of repeat units of thesaccharide, and the relative amount of free saccharide compared to thetotal amount of saccharide.

The oxo-eT linked glycoconjugates of the invention may be represented bythe general formula (I), as illustrated below:

wherein:A is a group (C═X)_(m) wherein X is S or O and m is 0 or 1;B is a bond, O, or CH₂; and when m is 0, B can also be (C═O);R is a C₂-C₁₆ alkylene, C₂-C₁₆ heteroakylene, NH—C(═O)—C₂-C₁₆ alkylene,or NH—C(═O)—C₂-C₁₆ heteroakylene, wherein said alkylene andheteroalkylene are optionally substituted by 1, 2 or 3 groupsindependently selected from COOR′ where R′ is selected from H, methyl,ethyl or propyl.

In the here above general formula (I), C₂-C₁₀ alkylene denotes astraight-chain or branched group containing 2 to 10 carbon atoms.Examples of suitable (C₂-C₁₀) alkylene radicals are (CH₂)₂, (CH₂)₃,CH₂—(CH—CH₃)—CH₂, (CH₂)₅, (CH₂)₆, (CH₂)₇, (CH₂)₈, (CH₂)₉, (CH₂)₁₀,(CH₂)₂—(CH—CH₃)—(CH₂)₂.

In the here above general formula (I), heteroalkylene refers to analkylene group as defined herein in which one or more of the carbonatoms is each independently replaced with the same or differentheteroatom selected from O, S or N. In a preferred embodiment,heteroalkylene refers to an alkylene group as defined herein in whichone or more of the carbon atoms is each replaced with an oxygen atom.Example of suitable C₂-C₂₀ heteroalkylene are O—CH₂, O—CH₂—CH₂,CH₂—CH₂—O—CH₂—CH₂, O—CH₂—CH₂—O—CH₂—CH₂, O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂,O—CH₂—CH₂—(N—CH₃)—CH₂—CH₂—O—CH₂—CH₂, CH₂—CH₂—(N—CH₃)—CH₂—CH₂,CH₂—CH₂—S—CH₂—CH₂ . . . .

In an embodiment, the oxo-eT linked glycoconjugates of the invention maybe represented by the general formula (I) above and wherein:

R═(CH₂)_(n) wherein n=1 to 10 orR═(CH₂CH₂O)_(n)CH₂CH₂ wherein n=1 to 5 orR═(CH(COOH)(CH₂)_(n) wherein n=1 to 10 orR═NHCO(CH₂)_(n) wherein n=1 to 10 orR═NHCO(CH₂CH₂O)_(n)CH₂ CH wherein n=1 to 4 orR═O(CH₂)_(n) wherein n=1 to 10 orR═O(CH₂CH₂O)_(n)CH₂CH wherein n=1 to 4.

The oxo-eTAC spacer provides stable carbamate, thioether and amide bondsbetween the saccharide and carrier protein. Synthesis of the oxo-eTAClinked glycoconjugate involves reaction of an activated hydroxyl groupof the saccharide with the amino group of a thioalkylamine reagent,forming a carbamate linkage to the saccharide to provide a thiolatedsaccharide. Generation of one or more free sulfhydryl groups isaccomplished by reaction with a reducing agent to provide an activatedthiolated saccharide (if thiol is protected). Reaction of the freesulfhydryl groups of the activated thiolated saccharide with anactivated carrier protein having one or more α-haloacetamide groups onamine containing residues generates a thioether bond to form theconjugate, wherein the carrier protein is attached to the oxo-eTACspacer through a thioether and an amide bond.

The oxo-eTAAN spacer, one of eT spacer's subgroup provides stable amine,thioether and amide bonds between the saccharide and carrier protein.The saccharide is first activated by oxidation of vicinal diol groupswith periodate or oxidation of primary hydroxyl groups with acombination of TEMPO and an oxidant to produce aldehyde groups.Oxidation of primary hydroxyl groups is preferred over periodateoxidsation since it does not involve chain scission thereby leading tominimal epitope modification. Synthesis of the oxo-eTAAN linkedglycoconjugate involves reaction of an activated saccharide aldehydewith the amino group of a bifunctional linker containing amine and thiolfunctionalities, forming an amine linkage to the saccharide to provide athiolated saccharide. Generation of one or more free sulfhydryl groupsis accomplished by reaction with a reducing agent to provide anactivated thiolated saccharide (if thiol is protected). Reaction of thefree sulfhydryl groups of the activated thiolated saccharide with anactivated carrier protein having one or more α-haloacetamide groups onamine containing residues generates a thioether bond to form theconjugate, wherein the carrier protein is attached to the oxo-eTAANspacer through a thioether and an amide bond.

The oxo-eTAAD spacer, one of oxo-eT spacer's subgroup provides stablethioether and amide bonds between the saccharide and carrier protein.Synthesis of the oxo-eTAAD linked glycoconjugate involves reaction of anactivated carboxyl group of the saccharide with the amino group of abifunctional linker containing amine and thiol functionalities, formingan amide linkage to the saccharide to provide a thiolated saccharide.Generation of one or more free sulfhydryl groups is accomplished byreaction with a reducing agent to provide an activated thiolatedsaccharide (if thiol is protected). Reaction of the free sulfhydrylgroups of the activated thiolated saccharide with an activated carrierprotein having one or more α-haloacetamide groups on amine containingresidues generates a thioether bond to form the conjugate, wherein thecarrier protein is attached to the oxo-eTAAD spacer through a thioetherand an amide bond.

In glycoconjugates of the invention, the saccharide may be apolysaccharide, an oligosaccharide, or a monosaccharide, and the carrierprotein may be selected from any suitable carrier as further describedherein or known to those of skill in the art. In frequent embodiments,the saccharide is a bacterial capsular polysaccharide. In some suchembodiments, the carrier protein is CRM₁₉₇.

In some such embodiments, the oxo-eT linked glycoconjugate comprises aPn capsular polysaccharide derived from S. pneumoniae. In specificembodiments, the Pn capsular polysaccharide is selected from the groupconsisting of Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F,14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides. Inother embodiments, the Pn capsular polysaccharide is selected from thegroup consisting of Pn-serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N,10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23B, 23F,33F and 35B capsular polysaccharides. In other embodiments, the Pncapsular polysaccharide is selected from the group consisting ofPn-serotype 2, 9N, 15A, 17F, 20, 23A, 23B and 35B capsularpolysaccharides. In other embodiments, the capsular polysaccharide isselected from the group consisting of Pn-Serotypes 10A, 11A, 22F and 33Fcapsular polysaccharides. In one such embodiment, the capsularpolysaccharide is a Pn-33F capsular polysaccharide. In another suchembodiment, the capsular polysaccharide is a Pn-22F capsularpolysaccharide. In another such embodiment, the capsular polysaccharideis a Pn-10A capsular polysaccharide. In yet another such embodiment, thecapsular polysaccharide is a Pn-11A capsular polysaccharide.

In other embodiments, the oxo-eT linked glycoconjugate comprises a Mncapsular polysaccharide derived from N. meningitidis. In specificembodiments, the Mn capsular polysaccharide is selected from the groupconsisting of Mn-serotype A, C, W135 and Y capsular polysaccharides. Inone such embodiment, the capsular polysaccharide is a Mn-A capsularpolysaccharide. In another such embodiment, the capsular polysaccharideis a Mn—C capsular polysaccharide. In another such embodiment, thecapsular polysaccharide is a Mn—W135 capsular polysaccharide. In yetanother such embodiment, the capsular polysaccharide is a Mn—Y capsularpolysaccharide. In another specific embodiment, the Mn capsularpolysaccharide is Mn-serotype X capsular polysaccharides.

In other embodiments, the oxo-eT linked glycoconjugate comprises a GBScapsular polysaccharide derived from Group B Streptococcus. In specificembodiments, the GBS capsular polysaccharide is selected from the groupconsisting of GBS-serotype Ia, Ib, II, III and V capsularpolysaccharides. In one such embodiment, the capsular polysaccharide isa GBS-Ia capsular polysaccharide. In another such embodiment, thecapsular polysaccharide is a GBS-Ib capsular polysaccharide. In anothersuch embodiment, the capsular polysaccharide is a GBS-II capsularpolysaccharide. In another such embodiment, the capsular polysaccharideis a GBS-III capsular polysaccharide. In yet another such embodiment,the capsular polysaccharide is a GBS-V capsular polysaccharide.

In other embodiments, the oxo-eT linked glycoconjugate comprises acapsular polysaccharide derived from Staphylococcus aureus. In specificembodiments, the S. aureus capsular polysaccharide is S. aureus serotype5 or 8 capsular polysaccharide.

In other embodiments, the oxo-eT linked glycoconjugate comprises acapsular polysaccharide derived from an Enterococcus bacteria. Inspecific embodiments, the Enterococcus capsular polysaccharide isEnterococcus faecalis or Enterococcus faecium capsular polysaccharide.

In particularly preferred embodiments, the oxo-eT linked glycoconjugatecomprises a Pn or Mn bacterial capsular polysaccharide, such as aPn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F or 33F capsular polysaccharide, or a Mn-serotype A,C, W135 or Y capsular polysaccharide, or a GBS-serotype Ia, Ib, II, IIIor V capsular polysaccharide, which is covalently conjugated to CRM₁₉₇through an oxo-eT spacer.

In other embodiments, the oxo-eT linked glycoconjugate comprises a S.aureus or Enterococcus bacterial capsular polysaccharide, such as S.aureus serotype 5 or 8 capsular polysaccharide, or Enterococcus faecalisor Enterococcus faecium capsular polysaccharide, which is covalentlyconjugated to CRM₁₉₇ through an oxo-eT spacer.

In some embodiments, the oxo-eT linked glycoconjugates of the presentinvention comprise a saccharide covalently conjugated to the carrierprotein through an oxo-eT spacer, wherein the saccharide has a molecularweight of between 10 kDa and 2,000 kDa. In other such embodiments, thesaccharide has a molecular weight of between 50 kDa and 2,000 kDa. Infurther such embodiments, the saccharide has a molecular weight ofbetween 50 kDa and 1,750 kDa; between 50 kDa and 1,500 kDa; between 50kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50 kDa and 750kDa; between 50 kDa and 500 kDa; between 100 kDa and 2,000 kDa; between100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDaand 1,250 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750kDa; between 100 kDa and 500 kDa; between 200 kDa and 2,000 kDa; between200 kDa and 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDaand 1,250 kDa; between 200 kDa and 1,000 kDa; between 200 kDa and 750kDa; or between 200 kDa and 500 kDa. In some such embodiments, thesaccharide is a bacterial capsular polysaccharide, such as a Pn-serotype1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F,22F, 23F or 33F capsular polysaccharide, or a Mn-serotype A, C, W135 orY capsular polysaccharide, or a GBS-serotype Ia, Ib, II, III or Vcapsular polysaccharide wherein the capsular polysaccharide has amolecular weight falling within any of the molecular weight ranges asdescribed. In some other embodiments, the saccharide is a bacterialcapsular polysaccharide, such as a capsular polysaccharide fromStaphylococcus aureus (e.g. S. aureus serotype 5 or 8 capsularpolysaccharide) or capsular polysaccharide from an Enterococcus bacteria(such as Enterococcus faecalis or Enterococcus faecium capsularpolysaccharide) wherein the capsular polysaccharide has a molecularweight falling within any of the molecular weight ranges as described.

In some embodiments, the oxo-eT linked glycoconjugate of the inventionhas a molecular weight of between 50 kDa and 20,000 kDa. In otherembodiments, the oxo-eT linked glycoconjugate has a molecular weight ofbetween 500 kDa and 10,000 kDa. In other embodiments, the oxo-eT linkedglycoconjugate has a molecular weight of between 200 kDa and 10,000 kDa.In still other embodiments, the oxo-eT linked glycoconjugate has amolecular weight of between 1,000 kDa and 3,000 kDa.

In further embodiments, the oxo-eT linked glycoconjugate of theinvention has a molecular weight of between 200 kDa and 20,000 kDa;between 200 kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between200 kDa and 7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDaand 3,000 kDa; between 200 kDa and 1,000 kDa; between 500 kDa and 20,000kDa; between 500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa;between 500 kDa and 10,000 kDa; between 500 kDa and 7,500 kDa; between500 kDa and 6,000 kDa; between 500 kDa and 5,000 kDa; between 500 kDaand 4,000 kDa; between 500 kDa and 3,000 kDa; between 500 kDa and 2,000kDa; between 500 kDa and 1,500 kDa; between 500 kDa and 1,000 kDa;between 750 kDa and 20,000 kDa; 750 kDa and 15,000 kDa; between 750 kDaand 12,500 kDa; between 750 kDa and 10,000 kDa; between 750 kDa and7,500 kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000 kDa;between 750 kDa and 4,000 kDa; between 750 kDa and 3,000 kDa; between750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between 1,000 kDaand 15,000 kDa; between 1,000 kDa and 12,500 kDa; between 1,000 kDa and10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa;between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 15,000 kDa;between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa;between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa;between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa; orbetween 2,000 kDa and 3,000 kDa.

Another way to characterize the oxo-eT linked glycoconjugates of theinvention is by the number of lysine residues in the carrier proteinthat become conjugated to the saccharide through an oxo-eT spacer, whichcan be characterized as a range of conjugated lysines.

In frequent embodiments, the carrier protein is covalently conjugated tothe oxo-eT spacer through an amide linkage to one or more ε-amino groupsof lysine residues on the carrier protein. In some such embodiments, thecarrier protein comprises 2 to 20 lysine residues covalently conjugatedto the saccharide. In other such embodiments, the carrier proteincomprises 4 to 16 lysine residues covalently conjugated to thesaccharide.

In a preferred embodiment, the carrier protein comprises CRM₁₉₇, whichcontains 39 lysine residues. In some such embodiments, the CRM₁₉₇ maycomprise 4 to 16 lysine residues out of 39 covalently linked to thesaccharide. Another way to express this parameter is that about 10% toabout 41% of CRM₁₉₇ lysines are covalently linked to the saccharide. Inanother such embodiment, the CRM₁₉₇ may comprise 2 to 20 lysine residuesout of 39 covalently linked to the saccharide. Another way to expressthis parameter is that about 5% to about 50% of CRM₁₉₇ lysines arecovalently linked to the saccharide.

The oxo-eT linked glycoconjugates of the invention may also becharacterized by the ratio (weight/weight) of saccharide to carrierprotein. In some embodiments, the saccharide: carrier protein ratio(w/w) is between 0.2 and 4. In other embodiments, the saccharide:carrier protein ratio (w/w) is between 1.0 and 2.5. In furtherembodiments, the saccharide: carrier protein ratio (w/w) is between 0.4and 1.7. In some such embodiments, saccharide is a bacterial capsularpolysaccharide, and/or the carrier protein is CRM₁₉₇.

Glycoconjugates may also be characterized by the number of covalentlinkages between the carrier protein and the saccharide as a function ofrepeat units of the saccharide. In one embodiment, the glycoconjugate ofthe invention comprises at least one covalent linkage between thecarrier protein and the polysaccharide for every 4 saccharide repeatunits of the polysaccharide. In another embodiment, the covalent linkagebetween the carrier protein and the polysaccharide occurs at least oncein every 10 saccharide repeat units of the polysaccharide. In anotherembodiment, the covalent linkage between the carrier protein and thepolysaccharide occurs at least once in every 15 saccharide repeat unitsof the polysaccharide. In a further embodiment, the covalent linkagebetween the carrier protein and the polysaccharide occurs at least oncein every 25 saccharide repeat units of the polysaccharide.

In frequent embodiments, the carrier protein is CRM₁₉₇ and the covalentlinkage via an oxo-eT spacer between the CRM₁₉₇ and the polysaccharideoccurs at least once in every 4, 10, 15 or 25 saccharide repeat units ofthe polysaccharide.

An important consideration during conjugation is the development ofconditions that permit the retention of potentially sensitivenon-saccharide substituent functional groups of the individualcomponents, such as O-Acyl, phosphate or glycerol phosphate side chainsthat may form part of the saccharide epitope.

In one embodiment, the glycoconjugate comprises a saccharide which has adegree of O-acetylation between 10-100%. In some such embodiments, thesaccharide has a degree of O-acetylation between 50-100%. In other suchembodiments, the saccharide has a degree of O-acetylation between75-100%. In further embodiments, the saccharide has a degree ofO-acetylation greater than or equal to 70% (≧70%).

The oxo-eT linked glycoconjugates and immunogenic compositions of theinvention may contain free saccharide that is not covalently conjugatedto the carrier protein, but is nevertheless present in theglycoconjugate composition. The free saccharide may be non-covalentlyassociated with (i.e., non-covalently bound to, adsorbed to, orentrapped in or with) the glycoconjugate.

In some embodiments, the oxo-eT linked glycoconjugate comprises lessthan about 45% free saccharide, less than about 40% free saccharide,less than about 35% free saccharide, less than about 30% freesaccharide, less than about 25% free saccharide, less than about 20%free saccharide, less than about 15% free saccharide, less than about10% free saccharide, or less than about 5% free saccharide relative tothe total amount of saccharide. Preferably, the glycoconjugate comprisesless than 15% free saccharide, more preferably less than 10% freesaccharide, and still more preferably, less than 5% of free saccharide.

In certain preferred embodiments, the invention provides an oxo-eTlinked glycoconjugate comprising a capsular polysaccharide, preferably aPn or Mn capsular polysaccharide, covalently conjugated to a carrierprotein through an oxo-eT spacer, having one or more of the followingfeatures alone or in combination: the polysaccharide has a molecularweight of between 50 kDa and 2,000 kDa; the glycoconjugate has amolecular weight of between 500 kDa to 10,000 KDa; the carrier proteincomprises 2 to 20 lysine residues covalently linked to the saccharide;the saccharide:carrier protein ratio (w/w) is between 0.2 and 4; theglycoconjugate comprises at least one covalent linkage between thecarrier protein and the polysaccharide for every 4, 10, 15 or 25saccharide repeat units of the polysaccharide; the saccharide has adegree of 0-acetylation between 75-100%; the conjugate comprises lessthan about 15% free polysaccharide relative to total polysaccharide; thecarrier protein is CRM₁₉₇; the capsular polysaccharide is selected fromPn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F or 33F capsular polysaccharides, or the capsularpolysaccharide is selected from Mn-serotype A, C, W135 or Y capsularpolysaccharides.

In certain preferred embodiments, the invention provides an oxo-eTlinked glycoconjugate comprising a capsular polysaccharide, preferably aPn or Mn capsular polysaccharide, covalently conjugated to a carrierprotein through an oxo-eT spacer, having one or more of the followingfeatures alone or in combination: the polysaccharide has a molecularweight of between 50 kDa and 2,000 kDa; the glycoconjugate has amolecular weight of between 500 kDa to 10,000 KDa; the carrier proteincomprises 2 to 20 lysine residues covalently linked to the saccharide;the saccharide:carrier protein ratio (w/w) is between 0.2 and 4; theglycoconjugate comprises at least one covalent linkage between thecarrier protein and the polysaccharide for every 4, 10, 15 or 25saccharide repeat units of the polysaccharide; the saccharide has adegree of 0-acetylation between 75-100%; the conjugate comprises lessthan about 15% free polysaccharide relative to total polysaccharide; thecarrier protein is CRM₁₉₇; the capsular polysaccharide is selected fromGBS-serotype Ia, Ib, II, III or V capsular polysaccharide or S. aureusserotype 5 or 8 capsular polysaccharide or Enterococcus faecalis orEnterococcus faecium capsular polysaccharide.

The oxo-eT linked glycoconjugates may also be characterized by theirmolecular size distribution (K_(d)). The molecular size of theconjugates is determined by Sepharose CL-4B stationary phase sizeexclusion chromatography (SEC) media using high pressure liquidchromatography system (HPLC). For K_(d) determination, thechromatography column is first calibrated to determine V₀, whichrepresents the void volume or total exclusion volume, and V_(i), thevolume at which the smallest molecules in the sample elutes, which isalso known as interparticle volume. All SEC separation takes placebetween V₀ and V_(i). The K_(d) value for each fraction collected isdetermined by the following expression K_(d)=(V_(e)−V_(i))/(V_(i)−V₀),where V_(e) represents the retention volume of the compound. The %fraction (major peak) that elutes ≦0.3 defines the conjugate K_(d)(molecular size distribution). In some embodiments, the inventionprovides oxo-eT linked glycoconjugates having a molecular sizedistribution (K_(d)) of ≧35%. In other embodiments, the inventionprovides oxo-eT linked glycoconjugates having a molecular sizedistribution (K_(d)) of ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%,≧60%, ≧70%, ≧80%, or ≧90%.

The oxo-eT linked glycoconjugates and immunogenic compositions of theinvention may contain free sulfhydryl residues. In some instances, theactivated thiolated saccharides formed by the methods provided hereinwill contain multiple free sulfhydryl residues, some of which may notundergo covalent conjugation to the carrier protein during theconjugation step. Such residual free sulfhydryl residues are capped byreaction with a thiol-reactive capping reagent, for exampleiodoacetamide (IAA), to cap the potentially reactive functionality.Other thiol-reactive capping reagents, e.g., maleimide containingreagents and the like, are also contemplated.

In addition, the oxo-eT linked glycoconjugates and immunogeniccompositions of the invention may contain residual unconjugated carrierprotein, which may include activated carrier protein which has undergonemodification during the capping process steps.

The glycoconjugates of the invention can be used in the production ofimmunogenic compositions to protect recipients from bacterialinfections, for example by pathogenic bacteria such as S. pneumonia orN. meningitidis or Group B Streptococcus or S. aureus or Enterococcusbacteria (such as Enterococcus faecalis or a Enterococcus faecium. Thus,in another aspect, the invention provides an immunogenic compositioncomprising an oxo-eT linked glycoconjugate and a pharmaceuticallyacceptable excipient, carrier or diluent, wherein the glycoconjugatecomprises a saccharide covalently conjugated to a carrier proteinthrough an oxo-eT spacer, as described herein.

In frequent embodiments, the immunogenic composition comprises an oxo-eTlinked glycoconjugate and a pharmaceutically acceptable excipient,carrier or diluent, wherein the glycoconjugate comprises a bacterialcapsular polysaccharide.

In some such embodiments, the immunogenic composition comprises anoxo-eT linked glycoconjugate which comprises a Pn capsularpolysaccharide derived from S. pneumoniae. In some specific embodiments,the Pn capsular polysaccharide is selected from the group consisting ofPn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C,19A, 19F, 22F, 23F and 33F capsular polysaccharides.

In other such embodiments, the immunogenic composition comprises anoxo-eT linked glycoconjugate which comprises a Mn capsularpolysaccharide derived from N. meningitidis. In some specificembodiments, the Mn capsular polysaccharide is selected from the groupconsisting of Mn-serotype A, C, W135 and Y capsular polysaccharides.

In other such embodiments, the immunogenic composition comprises anoxo-eT linked glycoconjugate which comprises a GBS capsularpolysaccharide derived from Group B Streptococcus. In some specificembodiments, the GBS capsular polysaccharide is selected from the groupconsisting of GBS-serotype Ia, Ib, II, III or V capsular polysaccharide.In other such embodiments, the immunogenic composition comprises anoxo-eT linked glycoconjugate which comprises a S. aureus capsularpolysaccharide. In some specific embodiments, the S. aureus capsularpolysaccharide is S. aureus serotype 5 or 8 capsular polysaccharide.

In other such embodiments, the immunogenic composition comprises anoxo-eT linked glycoconjugate which comprises an Enterococcus bacteriacapsular polysaccharide. In some specific embodiments, the Enterococcusbacteria capsular polysaccharide is Enterococcus faecalis orEnterococcus faecium capsular polysaccharide.

In particularly preferred embodiments, the immunogenic compositioncomprises an oxo-eT linked glycoconjugate which comprises a bacterialcapsular polysaccharide, such as a Pn or Mn or GBS capsularpolysaccharide, covalently conjugated to CRM₁₉₇ through an oxo-eTspacer.

In some embodiments, the immunogenic composition comprises an adjuvant.In some such embodiments, the adjuvant is an aluminum-based adjuvantselected from the group consisting of aluminum phosphate, aluminumsulfate and aluminum hydroxide. In one embodiment, the immunogeniccomposition comprises the adjuvant aluminum phosphate.

The oxo-eT linked glycoconjugates of the invention and immunogeniccompositions comprising them may contain some amount of free saccharide.In some embodiments, the immunogenic composition comprises less thanabout 45%, less than about 40%, less than about 35%, less than about30%, less than about 25%, less than about 20%, less than about 15%, lessthan about 10%, or less than about 5% free polysaccharide compared tothe total amount of polysaccharide. Preferably, the immunogeniccomposition comprises less than 15% free saccharide, more preferablyless than 10% free saccharide, and still more preferable, less than 5%of free saccharide.

In another aspect, the glycoconjugates or immunogenic compositions ofthe invention can be used to generate antibodies that are functional asmeasured by killing bacteria in an animal efficacy model or via anopsonophagocytic killing assay. Glycoconjugates of the inventioncomprising a bacterial capsular polysaccharide can be used in theproduction of antibodies against such a bacterial capsularpolysaccharide. Such antibodies subsequently can be used in research andclinical laboratory assays, such as bacterial detectiontion andserotyping. Such antibodies may also be used to confer passive immunityto a subject. In some embodiments, the antibodies produced againstbacterial polysaccharides are functional in either an animal efficacymodel or in an opsonophagocytic killing assay.

The oxo-eT linked glycoconjugates and immunogenic compositions describedherein may also be used in various therapeutic or prophylactic methodsfor preventing, treating or ameliorating a bacterial infection, diseaseor condition in a subject. In particular, oxo-eT linked glycoconjugatescomprising a bacterial antigen, such as a bacterial capsularpolysaccharide from a pathogenic bacteria, may be used to prevent, treator ameliorate a bacterial infection, disease or condition in a subjectcaused by pathogenic bacteria.

Thus in one aspect, the invention provides a method of preventing,treating or ameliorating a bacterial infection, disease or condition ina subject, comprising administering to the subject an immunologicallyeffective amount of an immunogenic composition of the invention, whereinsaid immunogenic composition comprises an oxo-eT linked glycoconjugatecomprising a bacterial capsular polysaccharide.

In one embodiment, the infection, disease or condition is associatedwith S. pneumonia bacteria and the glycoconjugate comprises a Pncapsular polysaccharide. In some such embodiments, the infection,disease or condition is selected from the group consisting of pneumonia,sinusitis, otitis media, meningitis, bacteremia, sepsis, pleuralempyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis,peritonitis, pericarditis, mastoiditis, cellulitis, soft tissueinfection and brain abscess.

In another embodiment, the infection, disease or condition is associatedwith N. meningitidis bacteria and the glycoconjugate comprises a Mncapsular polysaccharide. In some such embodiments, the infection,disease or condition is selected from the group consisting ofmeningitis, meningococcemia, bacteremia and sepsis.

In another embodiment, the infection, disease or condition is associatedwith Group B Streptococcus bacteria and the glycoconjugate comprises aGBS capsular polysaccharide.

In another aspect, the invention provides a method of inducing an immuneresponse in a subject, comprising administering to the subject animmunologically effective amount of an immunogenic compositioncomprising an oxo-eT linked glycoconjugate and a pharmaceuticallyacceptable excipient, carrier or diluent, wherein the glycoconjugatecomprises a bacterial capsular polysaccharide.

In yet another aspect, the invention provides a method for preventing,treating or ameliorating a disease or condition caused by pathogenicbacteria in a subject, comprising administering to the subject animmunologically effective amount of an immunogenic compositioncomprising an oxo-eT linked glycoconjugate and a pharmaceuticallyacceptable excipient, carrier or diluent, wherein the glycoconjugatecomprises a bacterial capsular polysaccharide.

In another aspect, the invention provides a method for reducing theseverity of at least one symptom of a disease or condition caused byinfection with pathogenic bacteria, comprising administering to asubject an immunologically effective amount of an immunogeniccomposition comprising an oxo-eT linked glycoconjugate and apharmaceutically acceptable excipient, carrier or diluent, wherein theglycoconjugate comprises a bacterial capsular polysaccharide, e.g., a Pnor Mn or GBS or S. aureus or Enterococcus capsular polysaccharide.

In another aspect, the invention provides a method of administering animmunologically effective amount of an immunogenic compositioncomprising an oxo-eT linked glycoconjugate of the invention to a subjectto generate a protective immune response in the subject, as furtherdescribed herein.

In yet another aspect, the invention provides an immunogenic compositioncomprising an oxo-eT linked glycoconjugate of the present invention, asdescribed herein, for use in the prevention, treatment or ameliorationof a bacterial infection, for example an infection by S. pneumonia or N.meningitidis or Group B streptococcus or S. aureus or Enterococcusbacteria (such as Enterococcus faecalis or Enterococcus faecium)

In another aspect, the invention provides the use of an immunogeniccomposition comprising an oxo-eT linked glycoconjugate of the presentinvention, as described herein, for the preparation of a medicament forthe prevention, treatment or amelioration of a bacterial infection, forexample infection by S. pneumonia or N. meningitidis or Group BStreptococcus or S. aureus or Enterococcus bacteria (such asEnterococcus faecalis or Enterococcus faecium).

In the therapeutic and/or prophylactic methods and uses described above,the immunogenic composition frequently comprises an oxo-eT linkedglycoconjugate comprising a bacterial capsular polysaccharide covalentlylinked to a carrier protein through an oxo-eT spacer. In frequentembodiments of the methods and described herein, the bacterial capsularpolysaccharide is a Pn capsular polysaccharide or a Mn capsularpolysaccharide or a GBS capsular polysaccharide or S. aureus capsularpolysaccharide or an Enterococcus bacteria capsular polysaccharide (suchas Enterococcus faecalis or Enterococcus faecium). In some suchembodiments, the capsular polysaccharide is selected from the groupconsisting of Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F,14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides. Inother such embodiments, the capsular polysaccharide is selected from thegroup consisting of Mn-serotype A, C, W135 and Y capsularpolysaccharides. In other such embodiments, the capsular polysaccharideis selected from the group consisting of GBS-serotype Ia, Ib, II, IIIand V capsular polysaccharides.

In other such embodiments, the capsular polysaccharide is selected, thecapsular polysaccharide is a S. aureus capsular polysaccharide. In othersuch embodiments, the S. aureus capsular polysaccharide is S. aureusserotype 5 or 8 capsular polysaccharide.

In other such embodiments, the capsular polysaccharide is anEnterococcus bacteria capsular polysaccharide. In other suchembodiments, the Enterococcus bacteria capsular polysaccharide isEnterococcus faecalis or Enterococcus faecium capsular polysaccharide.

In certain preferred embodiments, the carrier protein is CRM₁₉₇ Inparticularly preferred embodiments, the immunogenic compositioncomprises an oxo-eT linked glycoconjugate which comprises a bacterialcapsular polysaccharide, such as a Pn or Mn or GBS or S. aureus orEnterococcus bacteria capsular polysaccharide, covalently conjugated toCRM₁₉₇ through an oxo-eT spacer.

In addition, the present invention provides methods for inducing animmune response against S. pneumoniae or N. meningitidis or Group BStreptococcus or S. aureus or Enterococcus (such as Enterococcusfaecalis or a Enterococcus faecium) bacteria in a subject, methods forpreventing, treating or ameliorating an infection, disease or conditioncaused by S. pneumoniae or N. meningitidis bacteria or Group BStreptococcus or S. aureus or Enterococcus bacteria (such asEnterococcus faecalis or a Enterococcus faecium) in a subject, andmethods for reducing the severity of at least one symptom of aninfection, disease or condition caused by infection with S. pneumoniaeor N. meningitidis or Group B Streptococcus or S. aureus or Enterococcusbacteria (such as Enterococcus faecalis or a Enterococcus faecium) in asubject, in each case by administering to the subject an immunologicallyeffective amount of an immunogenic composition comprising an oxo-eTlinked glycoconjugate and a pharmaceutically acceptable excipient,carrier or diluent, wherein the glycoconjugate comprises a bacterialcapsular polysaccharide derived from S. pneumoniae or N. meningitidis orGroup B Streptococcus or S. aureus or Enterococcus bacteria (such asEnterococcus faecalis or a Enterococcus faecium), respectively.

Saccharides

Saccharides may include polysaccharides, oligosaccharides andmonosaccharides. In frequent embodiments, the saccharide is apolysaccharide, in particular a bacterial capsular polysaccharide.Capsular polysaccharides are prepared by standard techniques known tothose of ordinary skill in the art.

In the present invention, capsular polysaccharides may be prepared,e.g., from Pn-serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F,14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of S. pneumoniae. In oneembodiment, each pneumococcal polysaccharide serotype may be grown in asoy-based medium. Individual polysaccharides are purified throughcentrifugation, precipitation, ultra-filtration, and/or columnchromatography. Purified polysaccharides may be activated to make themcapable of reacting with the oxo-eT spacer and then incorporated intoglycoconjugates of the invention, as further described herein.

The molecular weight of the capsular polysaccharide is a considerationfor use in immunogenic compositions. High molecular weight capsularpolysaccharides are able to induce certain antibody immune responses dueto a higher valence of the epitopes present on the antigenic surface.The isolation and purification of high molecular weight capsularpolysaccharides is contemplated for use in the conjugates, compositionsand methods of the present invention.

In some embodiments, the saccharide has a molecular weight of between 10kDa and 2,000 kDa. In other such embodiments, the saccharide has amolecular weight of between 50 kDa and 2,000 kDa. In further suchembodiments, the saccharide has a molecular weight of between 50 kDa and1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa;between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDaand 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa;between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa;between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between200 kDa and 500 kDa. In some such embodiments, the saccharide is abacterial capsular polysaccharide, such as a Pn-serotype 1, 3, 4, 5, 6A,6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F or 33Fcapsular polysaccharide, or a Mn-serotype A, C, W135 or Y capsularpolysaccharide, or GBS-serotype Ia, Ib, II, III and V capsularpolysaccharides, or a S. aureus capsular polysaccharide or an orEnterococcus bacteria capsular polysaccharide (such as Enterococcusfaecalis or Enterococcus faecium) wherein the capsular polysaccharidehas a molecular weight falling within one of the molecular weight rangesas described.

In some embodiments, the saccharides of the invention are 0-acetylated.In some embodiments, the glycoconjugate comprises a saccharide which hasa degree of 0-acetylation of between 10-100%, between 20-100%, between30-100%, between 40-100%, between 50-100%, between 60-100%, between70-100%, between 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90% or80-90%. In other embodiments, the degree of 0-acetylation is ≧10%, ≧20%,≧30%, ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, or ≧90%, or about 100%.

In some embodiments, the capsular polysaccharides, glycoconjugates orimmunogenic compositions of the invention are used to generateantibodies that are functional as measured by the killing of bacteria inan animal efficacy model or an opsonophagocytic killing assay thatdemonstrates that the antibodies kill the bacteria.

Capsular polysaccharides can be obtained directly from bacteria usingisolation procedures known to one of ordinary skill in the art. See,e.g., Fournier et al. (1984), supra; Fournier et al. (1987) Ann. Inst.Pasteur/Microbiol. 138:561-567; US Patent Application Publication No.2007/0141077; and Intl Patent Application Publication No. WO 00/56357;each of which is incorporated herein by reference as if set forth in itsentirety). In addition, they can be produced using synthetic protocols.Moreover, capsular polysaccharide can be recombinantly produced usinggenetic engineering procedures also known to one of ordinary skill inthe art (see, Sau et al. (1997) Microbiology 143:2395-2405; and U.S.Pat. No. 6,027,925; each of which is incorporated herein by reference asif set forth in its entirety).

Bacterial strains of S. pneumoniae or N. meningitidis or Group BStreptoccus or S. aureus or Enterococcus bacteria (such as Enterococcusfaecalis or a Enterococcus faecium) used to make the respectivepolysaccharides that are used in the glycoconjugates of the inventionmay be obtained from established culture collections or clinicalspecimens.

Carrier Proteins

Another component of the glycoconjugate of the invention is a carrierprotein to which the saccharide is conjugated. The terms “proteincarrier” or “carrier protein” or “carrier” may be used interchangeablyherein. Carrier proteins are preferably proteins that are non-toxic andnon-reactogenic and obtainable in sufficient amount and purity. Carrierproteins should be amendable to standard conjugation procedures. In thenovel glycoconjugates of the invention, the carrier protein iscovalently linked to the saccharide through an oxo-eT spacer.

Conjugation to a carrier can enhance the immunogenicity of an antigen,for example bacterial antigen such as a bacterial capsularpolysaccharide. Preferred protein carriers for the antigens are toxins,toxoids or any mutant cross-reactive material (CRM) of the toxin fromtetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus andStreptococcus. In one embodiment, a particularly preferred carrier is ofdiphtheria toxoid CRM₁₉₇, derived from C. diphtheriae strain C7 (β197),which produces CRM₁₉₇ protein. This strain has ATCC accession No. 53281.A method for producing CRM₁₉₇ is described in U.S. Pat. No. 5,614,382,which is incorporated herein by reference as if set forth in itsentirety.

Alternatively, a fragment or epitope of the protein carrier or otherimmunogenic protein can be used. For example, a haptenic antigen can becoupled to a T-cell epitope of a bacterial toxin, toxoid or CRM. See, USPatent Application No. 150,688, filed Feb. 1, 1988, entitled “SyntheticPeptides Representing a T-Cell Epitope as a Carrier Molecule ForConjugate Vaccines”; incorporated herein by reference as if set forth inits entirety. Other suitable carrier proteins include inactivatedbacterial toxins such as cholera toxoid (e.g., as described in Int'lPatent Application No. WO 2004/083251), E. coli LT, E. coli ST, andexotoxin A from Pseudomonas aeruginosa. Bacterial outer membraneproteins such as outer membrane complex c (OMPC), porins, transferrinbinding proteins, pneumolysin, pneumococcal surface protein A (PspA),pneumococcal adhesion protein (PsaA) or Haemophilus influenzae protein Dalso can be used. Other proteins, such as ovalbumin, keyhole limpethemocyanin (KLH), bovine serum albumin (BSA) or purified proteinderivative of tuberculin (PPD) also can be used as carrier proteins.

In an embodiment, preferred carrier is selected in the group consistingof: DT (Diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT,CRM197 (a nontoxic but antigenically identical variant of diphtheriatoxin), other DT mutants (such as CRM176, CRM228, CRM 45 (Uchida et alJ. Biol. Chem. 218; 3838-3844, 1973), CRM9, CRM45, CRM102, CRM103 orCRM107; and other mutations described by Nicholls and Youle inGenetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992;deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat.No. 4,950,740; mutation of at least one or more residues Lys 516, Lys526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Pat.No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment disclosed in U.S.Pat. No. 5,843,711), pneumococcal pneumolysin (Kuo et al (1995) InfectImmun 63; 2706-13) including ply detoxified in some fashion for exampledPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX,including PhtA, PhtB, PhtD, PhtE (sequences of PhtA, PhtB, PhtD or PhtEare disclosed in WO 00/37105 or WO 00/39299) and fusions of Pht proteinsfor example PhtDE fusions, PhtBE fusions, Pht A-E (WO 01/98334, WO03/54007, WO2009/000826), OMPC (meningococcal outer membraneprotein—usually extracted from N. meningitidis serogroup B—EP0372501),PorB (from N. meningitidis), PD (Haemophilus influenzae protein D—see,e.g., EP 0 594 610 B), or immunologically functional equivalentsthereof, synthetic peptides (EP0378881, EP0427347), heat shock proteins(WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146),artificial proteins comprising multiple human CD4+ T cell epitopes fromvarious pathogen derived antigens (Falugi et al (2001) Eur J Immunol 31;3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72;4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptakeproteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761),transferrin binding proteins, pneumococcal adhesion protein (PsaA),recombinant Pseudomonas aeruginosa exotoxin A (in particular non-toxicmutants thereof (such as exotoxin A bearing a substution at glutamicacid 553 (Uchida Cameron D M, R J Collier. 1987. J. Bacteriol.169:4967-4971)). Other proteins, such as ovalbumin, keyhole limpethemocyanin (KLH), bovine serum albumin (BSA) or purified proteinderivative of tuberculin (PPD) also can be used as carrier proteins.Other suitable carrier proteins include inactivated bacterial toxinssuch as cholera toxoid (e.g., as described in Int'l Patent ApplicationNo. WO 2004/083251), E. coli LT, E. coli ST, and exotoxin A fromPseudomonas aeruginosa.

In an embodiment, the carrier is selected in the group consisting of TT,DT, DT mutants (such as CRM197), H. influenzae protein D, PhtX, PhtD,PhtDE fusions (particularly those described in WO 01/98334 and WO03/54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxinA or B of C. Difficile and PsaA.

In a preferred embodiment, the carrier protein of the glycoconjugates ofthe invention is DT (Diphtheria toxoid). In another embodiment, thecarrier protein of the glycoconjugates of the invention is TT (tetanustoxoid). In another embodiment, the carrier protein of theglycoconjugates of the invention is PD (Haemophilus influenzae proteinD—see, e.g., EP 0 594 610 B).

In a most preferred embodiment, the capsular saccharides of theinvention are conjugated to CRM₁₉₇ protein.

Accordingly, in frequent embodiments, the oxo-eT linked glycoconjugatescomprise CRM₁₉₇ as the carrier protein, wherein the capsularpolysaccharide is covalently linked to the oxo-eT spacer via a carbamatelinkage, and wherein the CRM₁₉₇ is covalently linked to the oxo-eTspacer via an amide linkage formed by an activated amino acid residue ofthe protein, typically through the ε-amine group of one or more lysineresidues.

The number of lysine residues in the carrier protein that becomeconjugated to the saccharide can be characterized as a range ofconjugated lysines. For example, in some embodiments of the immunogeniccompositions, the CRM₁₉₇ may comprise 4 to 16 lysine residues out of 39covalently linked to the saccharide. Another way to express thisparameter is that about 10% to about 41% of CRM₁₉₇ lysines arecovalently linked to the saccharide. In other embodiments, the CRM₁₉₇may comprise 2 to 20 lysine residues out of 39 covalently linked to thesaccharide. Another way to express this parameter is that about 5% toabout 50% of CRM₁₉₇ lysines are covalently linked to the saccharide.

The frequency of attachment of the saccharide chain to a lysine on thecarrier protein is another parameter for characterizing theglycoconjugates of the invention. For example, in some embodiments, atleast one covalent linkage between the carrier protein and thepolysaccharide for every 4 saccharide repeat units of thepolysaccharide. In another embodiment, the covalent linkage between thecarrier protein and the polysaccharide occurs at least once in every 10saccharide repeat units of the polysaccharide. In another embodiment,the covalent linkage between the carrier protein and the polysaccharideoccurs at least once in every 15 saccharide repeat units of thepolysaccharide. In a further embodiment, the covalent linkage betweenthe carrier protein and the polysaccharide occurs at least once in every25 saccharide repeat units of the polysaccharide.

In frequent embodiments, the carrier protein is CRM₁₉₇ and the covalentlinkage via an oxo-eT spacer between the CRM₁₉₇ and the polysaccharideoccurs at least once in every 4, 10, 15 or 25 saccharide repeat units ofthe polysaccharide. In some such embodiments, the polysaccharide is abacterial capsular polysaccharide derived from S. pneumoniae or N.meningitidis or Group B Streptococcus or a S. aureus polysaccharide oran Enterococcus bacteria polysaccharide (such as Enterococcus faecalisor Enterococcus faecium).

In other embodiments, the conjugate comprises at least one covalentlinkage between the carrier protein and saccharide for every 5 to 10saccharide repeat units; every 2 to 7 saccharide repeat units; every 3to 8 saccharide repeat units; every 4 to 9 saccharide repeat units;every 6 to 11 saccharide repeat units; every 7 to 12 saccharide repeatunits; every 8 to 13 saccharide repeat units; every 9 to 14 sacchariderepeat units; every 10 to 15 saccharide repeat units; every 2 to 6saccharide repeat units, every 3 to 7 saccharide repeat units; every 4to 8 saccharide repeat units; every 6 to 10 saccharide repeat units;every 7 to 11 saccharide repeat units; every 8 to 12 saccharide repeatunits; every 9 to 13 saccharide repeat units; every 10 to 14 sacchariderepeat units; every 10 to 20 saccharide repeat units; or every 4 to 25saccharide repeat units.

In another embodiment, at least one linkage between carrier protein andsaccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units ofthe polysaccharide.

Methods for Making Oxo-eT Linked Glycoconjugates Methods for MakingOxo-eTAC Linked Glycoconjugates

The present invention provides methods of making oxo-eTAC linkedglycoconjugates comprising a saccharide covalently conjugated to acarrier protein through a (((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC)spacer. The oxo-eTAC spacer, comprising stable carbamate, thioether andamide bonds, and serves to covalently link the saccharide and carrierprotein. One end of the oxo-eTAC spacer is covalently bound to ahydroxyl group of the saccharide through a carbamate linkage. The otherend of the oxo-eTAC spacer is covalently bound to an amino-containingresidue of the carrier protein, typically an ε-lysine residue, throughan amide linkage.

A representative route to the preparation of glycoconjugates of thepresent invention, comprising a polysaccharide conjugated to theactivated carrier protein CRM₁₉₇, is shown in FIG. 1.

Methods for Making Oxo-eTAAN Linked Glycoconjugates

The present invention provides methods of making oxo-eTAAN linkedglycoconjugates comprising a saccharide covalently conjugated to acarrier protein through a (((2-oxoethyl)thio)alkyl)amine (oxo-eTAAN)spacer. The oxo-eTAAN spacer contains stable amine, thioether and amidebonds, and serves to covalently link the saccharide and carrier protein.One end of the oxo-eTAAN spacer is covalently bound to a carbon atom ofthe saccharide through an amine linkage. The other end of the oxo-eTAANspacer is covalently bound to an amino-containing residue of the carrierprotein, typically an ε-lysine residue, through an amide linkage.

A representative route to the preparation of glycoconjugates of thepresent invention, comprising a polysaccharide conjugated to theactivated carrier protein CRM₁₉₇, is shown in FIG. 2.

Methods for Making Oxo-eTAAD Linked Glycoconjugates

The present invention provides methods of making oxo-eTAAD linkedglycoconjugates comprising a saccharide covalently conjugated to acarrier protein through a (((2-oxoethyl)thio)alkyl)amide (oxo-eTAAD)spacer. The oxo-eTAAD spacer contains stable amide, thioether and amidebonds, and serves to covalently link the saccharide and carrier protein.One end of the oxo-eTAAD spacer is covalently bound to a carboxyl groupof the saccharide through an amide linkage. The other end of theoxo-eTAAD spacer is covalently bound to an amino-containing residue ofthe carrier protein, typically an ε-lysine residue, through an amidelinkage.

A representative route to the preparation of glycoconjugates of thepresent invention, comprising a polysaccharide conjugated to theactivated carrier protein CRM₁₉₇, is shown in FIG. 3.

Methods for Making Oxo-eTAAD Linked Glycoconjugates Via ThiazolidinoneActivation Route

The present invention provides methods of making oxo-eTAAD linkedglycoconjugates comprising a saccharide covalently conjugated to acarrier protein through a (((2-oxoethyl)thio)alkyl)amide (oxo-eTAAD)spacer. The oxo-eTAAD spacer contains stable amide, thioether and amidebonds, and serves to covalently link the saccharide and carrier protein.One end of the oxo-eTAAD spacer is covalently bound to a carboxyl groupof the saccharide through an amide linkage. The other end of theoxo-eTAAD spacer is covalently bound to an amino-containing residue ofthe carrier protein, typically an ε-lysine residue, through an amidelinkage.

A representative route to the preparation of glycoconjugates of thepresent invention, via thiazolidinone activation, comprising apolysaccharide conjugated to the activated carrier protein CRM₁₉₇, isshown in FIG. 4.

The chemical structure of a representative bacterial capsularpolysaccharide, pneumococcal serotypes 33F, 10A, 11A and 22Fpolysaccharides derived from S. pneumoniae, having potential sites ofmodification using the oxo-eT spacer process are shown in FIG. 5, FIG.6, FIG. 7 and FIG. 8, respectively.

In one aspect, the method of making oxo-eTAC linked glycoconjugatecomprises the steps of: a) reacting a saccharide with a carbonic acidderivative, such as 1,1′-carbonyl-di-(1,2,4-triazole) (CDT) or1,1′-carbonyldiimidazole (CDI), in an organic solvent to produce anactivated saccharide; b) reacting the activated saccharide withheterobifunctional linker reagent having amine and thiol functionalitiesor a salt thereof, to produce a thiolated saccharide; c) reacting thethiolated saccharide with a reducing agent to produce an activatedthiolated saccharide comprising one or more free sulfhydryl residues; d)reacting the activated thiolated saccharide with an activated carrierprotein comprising one or more α-haloacetamide groups, to produce athiolated saccharide-carrier protein conjugate; and e) reacting thethiolated saccharide-carrier protein conjugate with (i) a first cappingreagent capable of capping unconjugated α-haloacetamide groups of theactivated carrier protein; and/or (ii) a second capping reagent capableof capping unconjugated free sulfhydryl residues of the activatedthiolated saccharide; whereby an oxo-eTAC linked glycoconjugate isproduced.

In a particularly preferred embodiment, the method comprises the stepsof: a) reacting a Pn-33F capsular polysaccharide with CDT or CDI in anorganic solvent to produce an activated Pn-33F polysaccharide; b)reacting the activated Pn-33F polysaccharide with a heterobifunctionallinker reagent having amine and thiol functionalities or a salt thereof,to produce a thiolated Pn-33F polysaccharide; c) reacting the thiolatedPn-33F polysaccharide with a reducing agent to produce an activatedthiolated Pn-33F polysaccharide comprising one or more free sulfhydrylresidues; d) reacting the activated thiolated Pn-33F polysaccharide withan activated CRM₁₉₇ carrier protein comprising one or moreα-bromoacetamide groups, to produce a thiolated Pn-33Fpolysaccharide-CRM₁₉₇ conjugate; and e) reacting the thiolated Pn-33Fpolysaccharide-CRM₁₉₇ conjugate with (i) N-acetyl-L-cysteine as a firstcapping reagent capable of capping unconjugated α-bromoacetamide groupsof the activated carrier protein; and (ii) iodoacetamide as a secondcapping reagent capable of capping unconjugated free sulfhydryl residuesof the activated thiolated Pn-33F polysaccharide; whereby an oxo-eTAClinked Pn-33F polysaccharide-CRM₁₉₇ glycoconjugate is produced.

In frequent embodiments, the carbonic acid derivative is CDT or CDI orDSC or N-hydroxysuccinimidyl chloroformate. Preferably the carbonic acidderivative is CDT, and the organic solvent is a polar aprotic solvent,such as dimethylsulfoxide (DMSO). Lyophilization of the activatedsaccharide is not required prior to the thiolation and/or conjugationsteps.

In a preferred embodiment, the thiolated saccharide is produced byreaction of the activated saccharide with the bifunctional symmetricthioalkylamine reagent, in the disulfide form, or a salt thereof. Apotential advantage to this reagent is that the symmetricalthioalkylamine linker, in the disulfide form, can react with twomolecules of activated saccharide, thus forming two molecules ofthiolated saccharide per molecule of thioalkylamine upon reduction ofthe disulfide bond. Alternatively, the thiolated saccharide may beformed by reaction of the activated saccharide with thioalkylamine inthe monomeric form, or a salt thereof. The oxo-eT linked glycoconjugatesproduced by the methods of the invention may be represented by generalformula (I).

In some embodiments of this aspect, step d) further comprises providingan activated carrier protein comprising one or more α-haloacetamidegroups, prior to reacting the activated thiolated saccharide with theactivated carrier protein, to produce a thiolated saccharide-carrierprotein conjugate. In frequent embodiments, the activated carrierprotein comprises one or more α-bromoacetamide groups.

The thiolated saccharide-carrier protein conjugate may be treated withone or more capping reagents capable of reacting with residual activatedfunctional groups present in the reaction mixture. Such residualreactive groups may be present on unreacted saccharide or carrierprotein components, due to incomplete conjugation or from the presenceof an excess of one of the components in the reaction mixture. In thatcase, capping may aid in the purification or isolation of theglycoconjugate. In some cases, residual activated functional groups maybe present in the glycoconjugate.

For example, excess α-haloacetamide groups on the activated carrierprotein may be capped by reaction with a low molecular weight thiol,such as N-acetyl-L-cysteine, which may be used in excess to ensurecomplete capping. Capping with N-acetyl-L-cysteine also permitsconfirmation of the conjugation efficiency, by detection of the uniqueamino acid S-carboxymethylcysteine (CMC) from the cysteine residues atthe capped sites, which can be determined by acidic hydrolysis and aminoacid analysis of the conjugation products. Detection of this amino acidconfirms successful capping of the reactive bromoacetamide groups, thusmaking them unavailable for any unwanted chemical reactions. Acceptablelevels of covalency and capping are between about 1-15 for CMCA/Lys andabout 0-5 for CMC/Lys. Similarly, excess free sulfhydryl residues can becapped by reaction with a low molecular weight electrophilic reagent,such as iodoacetamide. A portion of the CMCA may be derived from thepolysaccharide thiols capped directly by iodoacetamide that were notinvolved in conjugation with the haloacyl groups of the carrier protein.Therefore, post-conjugation reaction samples (prior to capping byiodoacetamide) need to be examined by amino acid analysis (CMCA) todetermine the accurate levels of thiols involved directly inconjugation. For a thiolated saccharide containing 10-12 thiols,typically 5-6 thiols are determined to be involved directly in theconjugation between the polysaccharide thiol and bromoacetylated proteinand 4-5 thiols are capped by iodoacetamide.

In preferred embodiments, the first capping reagent isN-acetyl-L-cysteine, which reacts with unconjugated α-haloacetamidegroups on the carrier protein. In other embodiments, the second cappingreagent is iodoacetamide (IAA), which reacts with unconjugated freesulfhydryl groups of the activated thiolated saccharide. Frequently,step e) comprises capping with N-acetyl-L-cysteine as the first cappingreagent and IAA as the second capping reagent. In some embodiments, thecapping step e) further comprises reaction with a reducing agent, forexample, DTT, TCEP, or mercaptoethanol, after reaction with the firstand/or second capping reagent.

In some embodiments, the method further comprises a step of purifyingthe oxo-eT linked glycoconjugate, for example, byultrafiltration/diafiltration.

In a preferred embodiment, the bifunctional symmetric thioalkylaminereagent is cystamine or a salt thereof is reacted with the activatedsaccharide to provide a thiolated saccharide or a salt thereof whichcontains a disulfide moiety.

Reaction of such thiolated saccharide derivatives with a reducing agentproduces an activated thiolated polysaccharide comprising one or morefree sulfhydryl residues (if thiol is protected). Such activatedthiolated saccharides can be isolated and purified, for example, byultrafiltration/diafiltration. Alternatively, the activated thiolatedsaccharides can be isolated and purified, for example, by standard sizeexclusion chromatographic (SEC) methods or ion exchange chromatographicmethods such as DEAE known in the art.

In the case of cystamine-derived, thiolated saccharides, reaction with areducing agent cleaves the disulfide bond to provide an activatedthiolated saccharide comprising one or more free sulfhydryl residues. Inthe case of cysteamine-derived, thiolated saccharides, reaction with areducing agent is optional and may be used to reduce disulfide bondsformed by oxidation of the reagent or product.

Reducing agents used in the methods of the invention include, forexample, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT) ormercaptoethanol. However, any suitable disulfide reducing agent may beused.

In some embodiments, the methods further comprise providing an activatedcarrier protein comprising one or more α-haloacetamide groups,preferably one or more α-bromoacetamide groups.

Reaction of the activated thiolated saccharide with an activated carrierprotein comprising one or more α-haloacetamide moieties results innucleophilic displacement of the α-halo group of the activated carrierprotein by the one or more free sulfhydryl groups of the activatedthiolated saccharide, forming the thioether bond of the oxo-eT spacer.

The α-haloacetylated amino acid residues of the carrier protein aretypically attached to the ε-amino groups of one or more lysine residuesof the carrier protein. In frequent embodiments, the carrier proteincontains one or more α-bromoacetylated amino acid residues. In oneembodiment, the carrier protein is activated with a bromoacetic acidreagent, such as the N-hydroxysuccinimide ester of bromoacetic acid(BAANS).

In one embodiment, the method includes the step of providing anactivated carrier protein comprising one or more α-haloacetamide groupsand reacting the activated thiolated polysaccharide with the activatedcarrier protein to produce a thiolated polysaccharide-carrier proteinconjugate, whereby a glycoconjugate comprising a polysaccharideconjugated to a carrier protein through an oxo-eT spacer is produced.

In some preferred embodiments of the methods herein, the bacterialcapsular polysaccharide is a Pn capsular polysaccharide derived from S.pneumoniae. In some such embodiments, the Pn capsular polysaccharide isselected from the group consisting of Pn-serotype 1, 3, 4, 5, 6A, 6B,7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33Fcapsular polysaccharides. In certain preferred embodiments, the carrierprotein is CRM₁₉₇ and the Pn capsular polysaccharide is selected fromthe group consisting of Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A,11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsularpolysaccharides.

In other preferred embodiments of the methods provided herein, thebacterial capsular polysaccharide is a Mn capsular polysaccharidederived from N. meningitidis. In some such embodiments, the Mn capsularpolysaccharide is selected from the group consisting of Mn-serotype A,C, W135 and Y capsular polysaccharides. In certain preferredembodiments, the carrier protein is CRM₁₉₇ and the capsularpolysaccharide is selected from the group consisting of Mn-serotype A,C, W135 and Y capsular polysaccharides.

In other preferred embodiments of the methods provided herein, thebacterial capsular polysaccharide is a GBS capsular polysaccharidederived from Group B Streptococcus. In some such embodiments, the GBScapsular polysaccharide is selected from the group consisting ofGBS-serotype Ia, Ib, II, III and V capsular polysaccharides. In certainpreferred embodiments, the carrier protein is CRM₁₉₇ and the capsularpolysaccharide is selected from the group consisting of GBS-serotype Ia,Ib, II, III and V capsular polysaccharides.

In some embodiments of each of methods provided herein, the saccharidewas compounded with imidazole or triazole and then reacted with acarbonic acid derivative, such as CDT, in an organic solvent (e.g.,DMSO) containing about 0.2% w/v water to produce activated saccharides.Use of the compounded saccharide in the activation step increases thesolubility of the saccharide in the organic solvent. Typically, thesaccharide was compounded with 10 grams of 1,2,4-triazole excipient pergram of polysaccharide followed by mixing at ambient temperature toprovide a compounded saccharide.

Thus, in certain embodiments the methods further comprise a step ofcompounding the saccharide with triazole or imidazole to give acompounded saccharide prior to the activation step a). In some suchembodiments, the compounded saccharide is shell-frozen, lyophilized andreconstituted in an organic solvent (such as DMSO) and about 0.2% w/vwater is added before activation with the carbonic acid derivative,e.g., CDT.

In one embodiment, the thiolated saccharide reaction mixture isoptionally treated with N-acetyl-lysine methyl ester to cap anyunreacted activated saccharide. In some such embodiments, the cappedthiolated saccharide mixture is purified byultrafiltration/diafiltration.

In frequent embodiments, the thiolated saccharide is reacted with areducing agent to produce an activated thiolated saccharide. In somesuch embodiments, the reducing agent is tris(-2-carboxyethyl)phosphine(TCEP), dithiothreitol (DTT) or mercaptoethanol. In some suchembodiments, the activated thiolated saccharide is purified byultrafiltration/diafiltration.

In one embodiment the method of producing an oxo-eT linkedglycoconjugate comprises the step of adjusting and maintaining the pH ofthe reaction mixture of activated thiolated saccharide and carrierprotein to a pH of about 8 to about 9 for about 20 hours at about 5° C.

In one embodiment, the method of producing a glycoconjugate of theinvention comprises the step of isolating the thiolatedsaccharide-carrier protein conjugate after it is produced. In frequentembodiments, the glycoconjugate is isolated byultrafiltration/diafiltration.

In another embodiment, the method of producing an oxo-eT linkedglycoconjugate of the invention comprises the step of isolating theisolated saccharide-carrier protein conjugate after it is produced. Infrequent embodiments, the glycoconjugate is isolated byultrafiltration/diafiltration.

In yet another embodiment, the method of producing the activatedsaccharide comprises the step of adjusting the water concentration ofthe reaction mixture comprising saccharide and CDT in an organic solventto between about 0.1 and 0.4%. In one embodiment, the waterconcentration of the reaction mixture comprising saccharide and CDT inan organic solvent is adjusted to about 0.2%.

In one embodiment, the step of activating the saccharide comprisesreacting the polysaccharide with an amount of CDT that is about a 5molar excess to the amount of polysaccharide present in the reactionmixture comprising capsular polysaccharide and CDT in an organicsolvent.

In another embodiment, the method of producing the glycoconjugate of theinvention comprises the step of determining the water concentration ofthe reaction mixture comprising saccharide. In one such embodiment, theamount of CDT added to the reaction mixture to activate the saccharideis provided in about an amount of CDT that is equimolar to the amount ofwater present in the reaction mixture comprising saccharide and CDT inan organic solvent.

In another embodiment, the amount of CDT added to the reaction mixtureto activate the saccharide is provided in about an amount of CDT that isat a molar ratio of about 0.5:1 compared to the amount of water presentin the reaction mixture comprising saccharide and CDT in an organicsolvent. In one embodiment, the amount of CDT added to the reactionmixture to activate the saccharide is provided in about an amount of CDTthat is at a molar ratio of 0.75:1 compared to the amount of waterpresent in the reaction mixture comprising saccharide and CDT in anorganic solvent.

In one embodiment, the method comprises the step of isolating thethiolated polysaccharide by diafiltration. In another embodiment, themethod comprises the step of isolating the activated thiolatedpolysaccharide by diafiltration.

In one embodiment, the carrier protein used in the method of producingan isolated Pn capsular polysaccharide-carrier protein conjugatecomprises CRM₁₉₇. In another embodiment, the carrier protein used in themethod of producing an isolated Mn capsular polysaccharide-carrierprotein conjugate comprises CRM₁₉₇.

In some embodiments, the saccharide:activated carrier protein ratio(w/w) is between 0.2 and 4. In other embodiments, thesaccharide:activated carrier protein ratio (w/w) is between 1.0 and 2.5.In further embodiments, the saccharide:activated carrier protein ratio(w/w) is between 0.4 and 1.7. In other embodiments, thesaccharide:activated carrier protein ratio (w/w) is about 1:1. In somesuch embodiments, the saccharide is a bacterial capsular polysaccharideand the activated carrier protein is generated by the activation(bromoacetylation) of CRM₁₉₇.

In another embodiment, the method of producing the activated saccharidecomprises the use of an organic solvent. In frequent embodiments, theorganic solvent is a polar aprotic solvent. In some such embodiments,the polar aprotic solvent is selected from the group consisting ofdimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide(DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile,1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) andhexamethylphosphoramide (HMPA), or a mixture thereof. In a preferredembodiment, the organic solvent is DMSO.

In frequent embodiments, isolation of the oxo-eT linked glycoconjugatecomprises a step of ultrafiltration/diafiltration.

In one embodiment, the saccharide used in the method of producing theglycoconjugate of the invention has a molecular weight between about 10kDa and about 2,000 kDa. In another embodiment, the saccharide used inthe method of producing the glycoconjugate of the invention has amolecular weight between about 50 kDa and about 2,000 kDa.

In one embodiment, glycoconjugate produced in the method of producingcapsular polysaccharide-carrier protein glycoconjugate has a sizebetween about between 50 kDa and about 20,000 kDa. In anotherembodiment, glycoconjugate produced in the method of producing capsularpolysaccharide-carrier protein glycoconjugate has a size between aboutbetween 500 kDa and about 10,000 kDa. In one embodiment, glycoconjugateproduced in the method of producing capsular polysaccharide-carrierprotein glycoconjugate has a size between about between 1,000 kDa andabout 3,000 kDa.

In another aspect, the invention provides an oxo-eT linkedglycoconjugate comprising a saccharide conjugated to a carrier proteinthrough an oxo-eT spacer, produced by any of the methods disclosedherein.

In another aspect, the invention provides an immunogenic compositioncomprising an oxo-eT linked glycoconjugate produced by any of themethods described herein.

The degree of 0-acetylation of the saccharide can be determined by anymethod known in the art, for example, by proton NMR (Lemercinier andJones (1996) Carbohydrate Research 296; 83-96, Jones and Lemercinier(2002) J. Pharmaceutical and Biomedical Analysis 30; 1233-1247, WO05/033148 or WO 00/56357). Another commonly used method is described byHestrin (1949) J. Biol. Chem. 180; 249-261. Yet another method is basedon HPLC-ion-exclusion chromatography. The degree of O-acetylation isdetermined by assessing the amount of free acetate present in a sampleand comparing that value to the amount of released acetate following amild base hydrolysis. Acetate is resolved from other components of thesample and quantitated with a Ultra-Violet (UV) detection at 210 nm.Another method is based on HPLC-ion-exclusion chromatography. O-Acetylis determined by assessing the amount of free acetate present in asample and comparing that value to the amount of released acetatefollowing a mild base hydrolysis. Acetate is resolved from othercomponents of the sample and quantitated with a Ultra-Violet (UV)detection at 210 nm.

Degree of Conjugation Determined by Amino Acid Analysis

Acid hydrolysis of the “pre-IAA capped” conjugate samples generatedusing bromoacetyl activation chemistry resulted in the formation of acidstable carboxymethylthioalkylamine (CMTA) from the conjugated sites andacid stable S-carboxymethylcysteine (CMC) from the cysteines at thecapped sites. Acid hydrolysis of the “post-IAA capped” conjugates(final) generated using the bromoacetyl activation chemistry alsoresulted in the formation of acid stable (CMTA) from the conjugatedsites and IAA capped sites as well and acid stableS-carboxymethylcysteine (CMC) from the cysteines at the capped sites.All of the unconjugated and uncapped lysines were converted back tolysine and detected as such. All other amino acids were hydrolyzed backto free amino acids except for tryptophan and cysteine, which weredestroyed by the hydrolysis conditions. Asparagine and glutamine wereconverted to aspartic acid and glutamic acid respectively.

The amino acids of each hydrolyzed sample and control were separatedusing ion exchange chromatography followed by reaction with BeckmanNinhydrin NinRX solution at 135° C. The derivatized amino acids werethen detected in the visible range at 570 nm and 440 nm (see Table 1). Astandard set of amino acids [Pierce Amino Acid Standard H] containing500 picomoles of each amino acid was run along with the samples andcontrols for each set of analysis. S-carboxymethylcysteine[Sigma-Aldrich] was added to the standard.

TABLE 1 Retention Times for Amino Acids Using Gradient Program 1 on theBeckman 6300 Amino Acid Analyzer WAVELENGTH RETENTION USED FOR TIME(MIN.) AMINO ACID DETECTION 8.3 Carboxymethylcysteine CMC 570 9.6Aspartic Acid & Asx 570 Asparagine 11.3 Threonine Thr 570 12.2 SerineSer 570 15.8 Glutamic Acid & Glx 570 & 440 Glutamine 18.5 Proline Pro440 21.8 Glycine Gly 570 23.3 Alanine Ala 570 29.0 Valine Val 570 32.8Methionine Met 570 35.5 Isoleucine Ile 570 36.8 Leucine Leu 570 40.5Tyrosine Tyr 570 42.3 Phenylalanine Phe 570 45.4 CarboxymethylcysteamineCMCA 570 48.8 Histidine His 570 53.6 Lysine Lys 570 70.8 Arginine Arg570

Lysine was chosen for the evaluation based on its covalent attachment toCysteine and Cysteamine and the expected similar hydrolysis. Theresulting numbers of moles of amino acids were then compared to theamino acid composition of the protein and reported along with the valuesfor CMC, CMTA or CMCA (in case of Cysteamine linker). The pre-IAA-CMTAvalue was used directly for evaluation of the degree of conjugation, theCMC value was used directly for evaluation of the degree of firstcapping and the Post-IAA-CMTA value was used for evaluation of thedegree of IAA (second) capping.

In one embodiment, the glycoconjugate is characterized by its molecularsize distribution (K_(d)). The molecular size of the conjugates isdetermined by Sepharose CL-4B stationary phase size exclusionchromatography (SEC) media using high pressure liquid chromatographysystem (HPLC). For K_(d) determination, the chromatography column isfirst calibrated to determine V₀, which represents the void volume ortotal exclusion volume and V_(i), the volume at which the smallestmolecules in the sample elute, also known as interparticle volume. AllSEC separation takes place between V₀ and V_(i). K_(d) value for eachfraction collected is determined by the following expressionK_(d)=(V_(e)−V_(i))/(V_(i)−V₀) where V_(e) represents the retentionvolume of the compound. The % fraction (major peak) that elutes ≦0.3defines the conjugate K_(d) (molecular size distribution).

Immunogenic Compositions

The term “immunogenic composition” relates to any pharmaceuticalcomposition containing an antigen (e.g., a microorganism or a componentthereof) which can be used to elicit an immune response in a subject.

As used herein, “immunogenic” means an ability of an antigen (or anepitope of the antigen), such as a bacterial capsular polysaccharide, ora glycoconjugate or immunogenic composition comprising a bacterialcapsular polysaccharide, to elicit an immune response in a host subject,such as a mammal, either humorally or cellularly mediated, or both.

The glycoconjugate may serve to sensitize the host by the presentationof the antigen in association with MHC molecules at a cell surface. Inaddition, antigen-specific T-cells or antibodies can be generated toallow for the future protection of an immunized host. Glycoconjugatesthus can protect the host from one or more symptoms associated withinfection by the bacteria, or may protect the host from death due to theinfection with the bacteria associated with the capsular polysaccharide.Glycoconjugates may also be used to generate polyclonal or monoclonalantibodies, which may be used to confer passive immunity to a subject.Glycoconjugates may also be used to generate antibodies that arefunctional as measured by the killing of bacteria in either an animalefficacy model or via an opsonophagocytic killing assay.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, unlessotherwise indicated by context, the term is intended to encompass notonly intact polyclonal or monoclonal antibodies, but also engineeredantibodies (e.g., chimeric, humanized and/or derivatized to altereffector functions, stability and other biological activities) andfragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (ScFv)and domain antibodies, including shark and camelid antibodies), andfusion proteins comprising an antibody portion, multivalent antibodies,multispecific antibodies (e.g., bispecific antibodies so long as theyexhibit the desired biological activity) and antibody fragments asdescribed herein, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site. Anantibody includes an antibody of any class, such as IgG, IgA, or IgM (orsub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantdomain of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 inhumans. The heavy-chain constant domains that correspond to thedifferent classes of immunoglobulins are called alpha, delta, epsilon,gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody.

The term “antigen” generally refers to a biological molecule, usually aprotein, peptide, polysaccharide or conjugate in an immunogeniccomposition, or immunogenic substance that can stimulate the productionof antibodies or T-cell responses, or both, in a subject, includingcompositions that are injected or absorbed into the subject. The immuneresponse may be generated to the whole molecule, or to a variousportions of the molecule (e.g., an epitope or hapten). The term may beused to refer to an individual molecule or to a homogeneous orheterogeneous population of antigenic molecules. An antigen isrecognized by antibodies, T-cell receptors or other elements of specifichumoral and/or cellular immunity. “Antigen” also includes all relatedantigenic epitopes. Epitopes of a given antigen can be identified usingany number of epitope mapping techniques, well known in the art. See,e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66(Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example,linear epitopes may be determined by, e.g., concurrently synthesizinglarge numbers of peptides on solid supports, the peptides correspondingto portions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002;Geysen et al. (1986) Molec. Immunol. 23:709-715; each of which isincorporated herein by reference as if set forth in its entirety.Similarly, conformational epitopes may be identified by determiningspatial conformation of amino acids such as by, e.g., x-raycrystallography and 2-dimensional nuclear magnetic resonance See, e.g.,Epitope Mapping Protocols, supra. Furthermore, for purposes of thepresent invention, “antigen” also can be used to refer to a protein thatincludes modifications, such as deletions, additions and substitutions(generally conservative in nature, but they may be non-conservative), tothe native sequence, as long as the protein maintains the ability toelicit an immunological response. These modifications may be deliberate,as through site-directed mutagenesis, or through particular syntheticprocedures, or through a genetic engineering approach, or may beaccidental, such as through mutations of hosts, which produce theantigens. Furthermore, the antigen can be derived, obtained, or isolatedfrom a microbe, e.g., a bacterium, or can be a whole organism.Similarly, an oligonucleotide or polynucleotide, which expresses anantigen, such as in nucleic acid immunization applications, is alsoincluded in the definition. Synthetic antigens are also included, e.g.,polyepitopes, flanking epitopes, and other recombinant or syntheticallyderived antigens (Bergmann et al. (1993) Eur. J. Immunol. 23:2777 2781;Bergmann et al. (1996) J. Immunol. 157:3242-3249; Suhrbier (1997)Immunol. Cell Biol. 75:402 408; Gardner et al. (1998) 12th World AIDSConference, Geneva, Switzerland, Jun. 28 to Jul. 3, 1998).

A “protective” immune response refers to the ability of an immunogeniccomposition to elicit an immune response, either humoral or cellmediated, or both, which serves to protect a subject from an infection.The protection provided need not be absolute, i.e., the infection neednot be totally prevented or eradicated, if there is a statisticallysignificant improvement compared with a control population of subjects,e.g. infected animals not administered the vaccine or immunogeniccomposition. Protection may be limited to mitigating the severity orrapidity of onset of symptoms of the infection. In general, a“protective immune response” would include the induction of an increasein antibody levels specific for a particular antigen in at least 50% ofsubjects, including some level of measurable functional antibodyresponses to each antigen. In particular situations, a “protectiveimmune response” could include the induction of a two fold increase inantibody levels or a fourfold increase in antibody levels specific for aparticular antigen in at least 50% of subjects, including some level ofmeasurable functional antibody responses to each antigen. In certainembodiments, opsonising antibodies correlate with a protective immuneresponse. Thus, protective immune response may be assayed by measuringthe percent decrease in the bacterial count in an opsonophagocytosisassay, for instance those described below. Preferably, there is adecrease in bacterial count of at least 10%, 25%, 50%, 65%, 75%, 80%,85%, 90%, 95% or more.

The terms an “immunogenic amount,” and an “immunologically effectiveamount,” which are used interchangeably herein, refers to the amount ofantigen or immunogenic composition sufficient to elicit an immuneresponse, which may be a cellular (T-cell) or humoral (B-cell orantibody) response, or both, where such an immune response may bemeasured by standard assays known to one skilled in the art. Typically,an immunologically effective amount will elicit a protective immuneresponse in a subject.

The immunogenic compositions of the present invention can be used toprophylactically or therapeutically, to protect or treat a subjectsusceptible to bacterial infection, e.g., by S. pneumonia or N.meningitidis or Group B Streptococcus or S. aureus or Enterococcusbacteria (such as Enterococcus faecalis or a Enterococcus faecium)bacteria, by means of administering the immunogenic compositions via asystemic, dermal or mucosal route, or can be used to generate apolyclonal or monoclonal antibody preparation that could be used toconfer passive immunity on another subject. These administrations caninclude injection via the intramuscular, intraperitoneal, intradermal orsubcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory or genitourinary tracts. Immunogeniccompositions may also be used to generate antibodies that are functionalas measured by the killing of bacteria in either an animal efficacymodel or via an opsonophagocytic killing assay.

Optimal amounts of components for a particular immunogenic compositioncan be ascertained by standard studies involving observation ofappropriate immune responses in subjects. Following an initialvaccination, subjects can receive one or several booster immunizationsadequately spaced.

In certain embodiments, the immunogenic composition will comprise one ormore adjuvants. As defined herein, an “adjuvant” is a substance thatserves to enhance the immunogenicity of an immunogenic composition ofthis invention. Thus, adjuvants are often given to boost the immuneresponse and are well known to the skilled artisan. Suitable adjuvantsto enhance effectiveness of the composition include, but are not limitedto:

-   -   (1) aluminum salts (alum), such as aluminum hydroxide, aluminum        phosphate, aluminum sulfate, etc.;    -   (2) oil-in-water emulsion formulations (with or without other        specific immunostimulating agents such as muramyl peptides        (defined below) or bacterial cell wall components), such as, for        example,        -   (a) MF59 (PCT Publ. No. WO 90/14837), containing 5%            Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally            containing various amounts of MTP-PE (see below, although            not required)) formulated into submicron particles using a            microfluidizer such as Model 110Y micro fluidizer            (Microfluidics, Newton, Mass.),        -   (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5%            pluronic-blocked polymer L121, and thr-MDP (see below)            either microfluidized into a submicron emulsion or vortexed            to generate a larger particle size emulsion, and        -   (c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, Mont.)            containing 2% Squalene, 0.2% Tween 80, and one or more            bacterial cell wall components from the group consisting of            3-O-deacylated monophosphorylipid A (MPL™) described in U.S.            Pat. No. 4,912,094 (Corixa), trehalose dimycolate (TDM), and            cell wall skeleton (CWS), preferably MPL+CWS (Detox™);    -   (3) saponin adjuvants, such as Quil A or STIMULON′ QS-21        (Antigenics, Framingham. Mass.) (U.S. Pat. No. 5,057,540) may be        used or particles generated therefrom such as ISCOMs        (immunostimulating complexes);    -   (4) bacterial lipopolysaccharides, synthetic lipid A analogs        such as aminoalkyl glucosamine phosphate compounds (AGP), or        derivatives or analogs thereof, which are available from Corixa,        and which are described in U.S. Pat. No. 6,113,918; one such AGP        is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl        2-Deoxy-4-O-phosphono-3-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside,        which is also known as 529 (formerly known as RC529), which is        formulated as an aqueous form or as a stable emulsion, synthetic        polynucleotides such as oligonucleotides containing CpG motif(s)        (U.S. Pat. No. 6,207,646);    -   (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4,        IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g.,        gamma interferon), granulocyte macrophage colony stimulating        factor (GM-CSF), macrophage colony stimulating factor (M-CSF),        tumor necrosis factor (TNF), costimulatory molecules B7-1 and        B7-2. etc.;    -   (6) detoxified mutants of a bacterial ADP-ribosylating toxin        such as a cholera toxin (CT) either in a wild-type or mutant        form, for example, where the glutamic acid at amino acid        position 29 is replaced by another amino acid, preferably a        histidine, in accordance with published international patent        application number WO 00/18434 (see also WO 02/098368 and WO        02/098369), a pertussis toxin (PT), or an E. coli heat-labile        toxin (LT), particularly LT-K63, LT-R72, CT-5109, PT-K9/G129        (see, e.g., WO 93/13302 and WO 92/19265); and    -   (7) other substances that act as immunostimulating agents to        enhance the effectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetylnormuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In certain embodiments, the adjuvant is an aluminum-based adjuvant, suchas an aluminum salt. In specific embodiments, the aluminum-basedadjuvant is selected from the group consisting of aluminum phosphate,aluminum sulfate and aluminum hydroxide. In a specific embodiment, theadjuvant is aluminum phosphate.

The immunogenic composition optionally can comprise a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers includecarriers approved by a regulatory agency of a Federal, a stategovernment, or other regulatory agency, or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use insubjects, including humans as well as non-human mammals. The termcarrier may be used to refer to a diluent, excipient, or vehicle withwhich the pharmaceutical composition is administered. Water, salinesolutions and aqueous dextrose and glycerol solutions can be employed asliquid carriers, particularly for injectable solutions. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. The formulation should suitthe mode of administration.

The immunogenic compositions of the invention may further comprise oneor more preservatives in addition to a plurality of capsularpolysaccharide-protein conjugates. The FDA requires that biologicalproducts in multiple-dose (multi-dose) vials contain a preservative,with only a few exceptions. Vaccine products containing preservativesinclude vaccines containing benzethonium chloride (anthrax),2-phenoxyethanol (DTaP, HepA, Lyme, Polio (parenteral)), phenol (Pneumo,Typhoid (parenteral), Vaccinia) and thimerosal (DTaP, DT, Td, HepB, Hib,Influenza, JE, Mening, Pneumo, Rabies). Preservatives approved for usein injectable drugs include, e.g., chlorobutanol, m-cresol,methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride,benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosaland phenylmercuric nitrate.

In certain embodiments, a formulation of the invention which iscompatible with parenteral administration comprises one or morenon-ionic surfactants, including but not limited to polyoxyethylenesorbitan fatty acid esters, Polysorbate-80 (Tween 80), Polysorbate-60(Tween 60), Polysorbate-40 (Tween 40) and Polysorbate-20 (Tween 20),polyoxyethylene alkyl ethers, including but not limited to Brij 58, Brij35, as well as others such as Triton X-100; Triton X-114, NP40, Span 85and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121),with preferred components Polysorbate-80 at a concentration from about0.001% to about 2% (with up to about 0.25% being preferred) orPolysorbate-40 at a concentration from about 0.001% to 1% (with up toabout 0.5% being preferred).

Packaging and Dosing Forms

Direct delivery of immunogenic compositions of the present invention toa subject may be accomplished by parenteral administration(intramuscularly, intraperitoneally, intradermally, subcutaneously,intravenously, or to the interstitial space of a tissue); or via mucosaladministration to the oral/alimentary, respiratory or genitourinarytracts; or by topical, transdermal, intranasal, ocular, aural, pulmonaryor other mucosal administration.

In one embodiment, parenteral administration is by intramuscularinjection, e.g., to the thigh or upper arm of the subject. Injection maybe via a needle (e.g. a hypodermic needle), but needle free injectionmay alternatively be used. A typical intramuscular dose is 0.5 mL. Inanother embodiment, intranasal administration is used for the treatmentof pneumonia or otitis media (as nasopharyngeal carriage of pneumococcican be more effectively prevented, thus attenuating infection at itsearliest stage).

Compositions of the invention may be prepared in various forms, e.g.,for injection either as liquid solutions or suspensions. In certainembodiments, the composition may be prepared as a powder or spray forpulmonary administration, e.g. in an inhaler. In other embodiments, thecomposition may be prepared as a suppository or pessary, or for nasal,aural or ocular administration, e.g., as a spray, drops, gel or powder.

The amount of glycoconjugate in each immunogenic composition dose isselected as an amount that induces an immunoprotective response withoutsignificant, adverse effects. Such amount can vary depending upon thebacterial serotype present in the glycoconjugate. Generally, each dosewill comprise 0.1 to 100 μg of polysaccharide, particularly 0.1 to 10μg, and more particularly 1 to 5 μg.

In a particular embodiment of the present invention, the immunogeniccomposition is a sterile liquid formulation of a Pn or Mn capsularpolysaccharide individually conjugated to CRM₁₉₇ via an oxo-eT linker,wherein each 0.5 mL dose is formulated to contain 1-5 μg ofpolysaccharide, which may further contain 0.125 mg of elemental aluminum(0.5 mg aluminum phosphate) adjuvant; and sodium chloride and sodiumsuccinate buffer as excipients.

Optimal amounts of components for a particular immunogenic compositionmay be ascertained by standard studies involving observation ofappropriate immune responses in subjects. Following an initialvaccination, subjects can receive one or several booster immunizationsadequately spaced.

Immunogenic compositions of the invention may be packaged in unit doseor multi-dose form (e.g., 2 doses, 4 doses, or more). For multi-doseforms, vials are typically but not necessarily preferred over pre-filledsyringes. Suitable multi-dose formats include but are not limited to: 2to 10 doses per container at 0.1 to 2 mL per dose. In certainembodiments, the dose is a 0.5 mL dose. See, e.g., International PatentApplication WO 2007/127668, which is incorporated by reference herein.

Compositions may be presented in vials or other suitable storagecontainers, or may be presented in pre-filled delivery devices, e.g.,single or multiple component syringes, which may be supplied with orwithout needles. A syringe typically but need not necessarily contains asingle dose of the preservative-containing immunogenic composition ofthe invention, although multi-dose, pre-filled syringes are alsoenvisioned. Likewise, a vial may include a single dose but mayalternatively include multiple doses.

Effective dosage volumes can be routinely established, but a typicaldose of the composition for injection has a volume of 0.5 mL. In certainembodiments, the dose is formulated for administration to a humansubject. In certain embodiments, the dose is formulated foradministration to an adult, teen, adolescent, toddler or infant (i.e.,no more than one year old) human subject and may in preferredembodiments be administered by injection.

Liquid immunogenic compositions of the invention are also suitable forreconstituting other immunogenic compositions which are presented inlyophilized form. Where an immunogenic composition is to be used forsuch extemporaneous reconstitution, the invention provides a kit withtwo or more vials, two or more ready-filled syringes, or one or more ofeach, with the contents of the syringe being used to reconstitute thecontents of the vial prior to injection, or vice versa.

In yet another embodiment, a container of the multi-dose format isselected from one or more of the group consisting of, but not limitedto, general laboratory glassware, flasks, beakers, graduated cylinders,fermentors, bioreactors, tubings, pipes, bags, jars, vials, vialclosures (e.g., a rubber stopper, a screw on cap), ampoules, syringes,dual or multi-chamber syringes, syringe stoppers, syringe plungers,rubber closures, plastic closures, glass closures, cartridges anddisposable pens and the like. The container of the present invention isnot limited by material of manufacture, and includes materials such asglass, metals (e.g., steel, stainless steel, aluminum, etc.) andpolymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers).In a particular embodiment, the container of the format is a 5 mL SchottType 1 glass vial with a butyl stopper. The skilled artisan willappreciate that the format set forth above is by no means an exhaustivelist, but merely serve as guidance to the artisan with respect to thevariety of formats available for the present invention. Additionalformats contemplated for use in the present invention may be found inpublished catalogues from laboratory equipment vendors and manufacturerssuch as United States Plastic Corp. (Lima, Ohio), VWR.

Methods for Inducing an Immune Response and Protecting Against Infection

The present invention also includes methods for using oxo-eT linkedglycoconjugates and immunogenic compositions comprising them, eitherprophylactically or therapeutically. For example, one aspect of theinvention provides a method of inducing an immune response against apathogenic bacteria, for example pneumococcal or meningococcal bacteria,comprising administering to a subject an immunologically effectiveamount of any of the immunogenic compositions described hereincomprising a bacterial antigen, such as a bacterial capsularpolysaccharide derived from pathogenic bacteria. One embodiment of theinvention provides a method of protecting a subject against an infectionby pathogenic bacteria, or a method of preventing, treating orameliorating an infection disease or condition associated with apathogenic bacteria, or a method of reducing the severity of or delayingthe onset of at least one symptom associated with an infection caused bypathogenic bacteria, in each case the methods comprising administeringto a subject an immunologically effective amount of any of theimmunogenic compositions described herein comprising a bacterialantigen, such as a bacterial capsular polysaccharide derived from thepathogenic bacteria.

One embodiment of the invention provides a method of preventing,treating or ameliorating a bacterial infection, disease or condition ina subject, comprising administering to the subject an immunologicallyeffective amount of an immunogenic composition of the invention, whereinsaid immunogenic composition comprises an oxo-eT linked glycoconjugatecomprising a bacterial antigen, such as a bacterial capsularpolysaccharide.

In some embodiments, the method of preventing, treating or amelioratinga bacterial infection, disease or condition comprises human, veterinary,animal, or agricultural treatment. Another embodiment provides a methodof preventing, treating or ameliorating a bacterial infection, diseaseor condition associated with pathogenic bacteria in a subject, themethod comprising generating a polyclonal or monoclonal antibodypreparation from the immunogenic composition described herein, and usingsaid antibody preparation to confer passive immunity to the subject. Oneembodiment of the invention provides a method of preventing a bacterialinfection in a subject undergoing a surgical procedure, the methodcomprising the step of administering a prophylactically effective amountof an immunogenic composition described herein to the subject prior tothe surgical procedure.

In preferred embodiments of each of the foregoing methods, thepathogenic bacteria are pneumococcal or meningococcal bacteria, such asS. pneumoniae or N. meningitis bacteria. In some such embodiments, thebacterial antigen is a capsular polysaccharide selected from the groupconsisting of Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F,14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides. Inother such embodiments, the bacterial antigen is a capsularpolysaccharide selected from the group consisting of Mn-serotype A, C,W135 and Y capsular polysaccharides. In other such embodiments, thebacterial antigen is a capsular polysaccharide selected from the groupconsisting of GBS-serotype Ia, Ib, II, III and V capsularpolysaccharides. In other such embodiments, the capsular polysaccharideis a capsular polysaccharide from Staphylococcus aureus (e.g. S. aureusserotype 5 or 8 capsular polysaccharide) or a capsular polysaccharidefrom an Enterococcus bacteria (such as Enterococcus faecalis orEnterococcus faecium capsular polysaccharide).

An immune response to an antigen or immunogenic composition ischaracterized by the development in a subject of a humoral and/or acell-mediated immune response to molecules present in the antigen orimmunogenic composition of interest. For purposes of the presentinvention, a “humoral immune response” is an antibody-mediated immuneresponse and involves the induction and generation of antibodies thatrecognize and bind with some affinity for the antigen in the immunogeniccomposition of the invention, while a “cell-mediated immune response” isone mediated by T-cells and/or other white blood cells. A “cell-mediatedimmune response” is elicited by the presentation of antigenic epitopesin association with Class I or Class II molecules of the majorhistocompatibility complex (MHC), CD1 or other non-classical MHC-likemolecules. This activates antigen-specific CD4+ T helper cells or CD8+cytotoxic T lymphocyte cells (“CTLs”). CTLs have specificity for peptideantigens that are presented in association with proteins encoded byclassical or non-classical MHCs and expressed on the surfaces of cells.CTLs help induce and promote the intracellular destruction ofintracellular microbes, or the lysis of cells infected with suchmicrobes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide or other antigens in associationwith classical or non-classical MHC molecules on their surface. A“cell-mediated immune response” also refers to the production ofcytokines, chemokines and other such molecules produced by activatedT-cells and/or other white blood cells, including those derived fromCD4+ and CD8+ T-cells. The ability of a particular antigen orcomposition to stimulate a cell-mediated immunological response may bedetermined by a number of assays, such as by lymphoproliferation(lymphocyte activation) assays, CTL cytotoxic cell assays, by assayingfor T-lymphocytes specific for the antigen in a sensitized subject, orby measurement of cytokine production by T cells in response tore-stimulation with antigen. Such assays are well known in the art. See,e.g., Erickson et al. (1993) J. Immunol. 151:4189-4199; and Doe et al.(1994) Eur. J. Immunol. 24:2369-2376.

The immunogenic compositions and methods of the invention may be usefulfor one or more of the following: (i) the prevention of infection orre-infection, as in a traditional vaccine, (ii) the reduction in theseverity of, or, in the elimination of symptoms, and/or (iii) thesubstantial or complete elimination of the pathogen or disorder inquestion. Hence, treatment may be effected prophylactically (prior toinfection) or therapeutically (following infection). In the presentinvention, prophylactic treatment is the preferred mode. According to aparticular embodiment of the present invention, compositions and methodsare provided that treat, including prophylactically and/ortherapeutically immunize, a host subject against bacterial infection,e.g., by S. pneumoniae or N. meningitidis, GBS or S. aureus orEnterococcus bacteria (such as Enterococcus faecalis or a Enterococcusfaecium. The methods of the present invention are useful for conferringprophylactic and/or therapeutic immunity to a subject. The methods ofthe present invention can also be practiced on subjects for biomedicalresearch applications.

As used herein, the term “subject” means a human or non-human animal.More particularly, subject refers to any animal classified as a mammal,including humans, domestic and farm animals, and research, zoo, sportsand pet companion animals such as a household pet and other domesticatedanimals including, but not limited to, cattle, sheep, ferrets, swine,horses, rabbits, goats, dogs, cats, and the like. Preferred companionanimals are dogs and cats. Preferably, the subject is human.

The amount of a particular conjugate in a composition is generallycalculated based on total amount of polysaccharide, both conjugated andnon-conjugated for that conjugate. For example, a conjugate with 20%free polysaccharide will have about 80 μg of conjugated polysaccharideand about 20 μg of non-conjugated polysaccharide in a 100 μgpolysaccharide dose. The protein contribution to the conjugate isusually not considered when calculating the dose of a conjugate. Theimmunogenic amount of a conjugate or immunogenic composition may varydepending upon the bacterial serotype. Generally, each dose willcomprise 0.1 to 100 μg of polysaccharide, particularly 0.1 to 10 μg, andmore particularly 1 to 10 μg. The immunogenic amount of the differentpolysaccharide components in an immunogenic composition may diverge andeach may comprise 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg,10 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, orabout 100 μg of any particular polysaccharide antigen.

The term “invasive disease” refers to the isolation of bacteria from anormally sterile site, where there are associated clinicalsigns/symptoms of disease. Normally sterile body sites include blood,CSF, pleural fluid, pericardial fluid, peritoneal fluid, joint/synovialfluid, bone, internal body site (lymph node, brain, heart, liver,spleen, vitreous fluid, kidney, pancreas, ovary) or other normallysterile sites. Clinical conditions characterizing invasive diseasesinclude bacteremia, pneumonia, cellulitis, osteomyelitis, endocarditis,septic shock and more.

The effectiveness of an antigen as an immunogen can be measured eitherby proliferation assays, by cytolytic assays, such as chromium releaseassays to measure the ability of a T-cell to lyse its specific targetcell, or by measuring the levels of B-cell activity by measuring thelevels of circulating antibodies specific for the antigen in serum. Animmune response may also be detected by measuring the serum levels ofantigen specific antibody induced following administration of theantigen, and more specifically, by measuring the ability of theantibodies so induced to enhance the opsonophagocytic ability ofparticular white blood cells, as described herein. The level ofprotection of the immune response may be measured by challenging theimmunized host with the antigen that has been administered. For example,if the antigen to which an immune response is desired is a bacterium,the level of protection induced by the immunogenic amount of the antigenis measured by detecting the percent survival or the percent mortalityafter challenge of the animals with the bacterial cells. In oneembodiment, the amount of protection may be measured by measuring atleast one symptom associated with the bacterial infection, e.g., a feverassociated with the infection. The amount of each of the antigens in themulti-antigen or multi-component vaccine or immunogenic compositionswill vary with respect to each of the other components and can bedetermined by methods known to the skilled artisan. Such methods wouldinclude procedures for measuring immunogenicity and/or in vivo efficacy.

In another aspect, the invention provides antibodies and antibodycompositions which bind specifically and selectively to the capsularpolysaccharides or glycoconjugates of the present invention. In somesuch embodiments, the invention provides antibodies and antibodycompositions which bind specifically and selectively to the Pn-serotype1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F,22F, 23F or 33F capsular polysaccharides or glycoconjugates comprisingthem. In other such embodiments, the invention provides antibodies andantibody compositions which bind specifically and selectively to theMn-serotype A, C, W135 or Y capsular polysaccharides or glycoconjugatescomprising them. In other such embodiments, the invention providesantibodies and antibody compositions which bind specifically andselectively to the GBS-serotype Ia, Ib, II, III and V capsularpolysaccharides or glycoconjugates comprising them. In other suchembodiments, the invention provides antibodies and antibody compositionswhich bind specifically and selectively to the S. aureus serotype 5 or 8capsular polysaccharide or Enterococcus faecalis or Enterococcus faeciumcapsular polysaccharides or glycoconjugates comprising them. In someembodiments, antibodies are generated upon administration to a subjectof the capsular polysaccharides or glycoconjugates of the presentinvention. In some embodiments, the invention provides purified orisolated antibodies directed against one or more of the capsularpolysaccharides or glycoconjugates of the present invention. In someembodiments, the antibodies of the present invention are functional asmeasured by killing bacteria in either an animal efficacy model or viaan opsonophagocytic killing assay. Antibodies or antibody compositionsof the invention may be used in a method of treating or preventing abacterial infection, disease or condition associated with pathogenicbacteria in a subject, e.g., S. pneumoniae or N. meningitidis or Group BStreptoccus or S. aureus or Enterococcus bacteria, the method comprisinggenerating a polyclonal or monoclonal antibody preparation, and usingsaid antibody or antibody composition to confer passive immunity to thesubject. Antibodies of the invention may also be useful for diagnosticmethods, e.g., detecting the presence of or quantifying the levels ofcapsular polysaccharide or a glycoconjugate thereof. For example,antibodies of the invention may also be useful for detecting thepresence of or quantifying the levels of a Pn or Mn or GBS or S. aureusor Enterococcus capsular polysaccharide or a glycoconjugate thereof,wherein the glycoconjugate comprises the bacterial capsularpolysaccharide conjugated to a carrier protein through an oxo-eT spacer.

Several assays and animal models known in the art may be used to assessthe efficacy of any one of the immunogenic compositions describedherein. For example, Chiavolini et al. Clin. Microbiol. Rev. (2008),21(4):666-685) describe animal models of S. pneumoniae diseases.Gorringe et al. METHODS IN MOLECULAR MEDICINE, vol. 66 (2001), Chapter17, Pollard and Maiden eds. (Humana Press Inc.) describe animal modelsfor meningococcal diseases.

Opsonophagocytic Activity (OPA) Assay

OPA assay procedures were based on the methods previously described byHu, et al. (Clin. Diagn. Lab. Immunol. 2005; 12(2):287-95), with thefollowing modifications. Heat-inactivated sera were serially diluted2.5-fold in buffer. Target bacteria were added to assay plates and wereincubated for 30 min at 25° C. on a shaker. Baby rabbit complement (3-to 4-weekold, Pel-Freez, 12.5% final concentration) and differentiatedHL-60 cells, were then added to each well at an approximate effector totarget ratio of 200:1. Assay plates were incubated for 45 min at 37° C.on a shaker. To terminate the reaction, 80 μL of 0.9% NaCl was added toall wells, mixed, and a 10-4 aliquot were transferred to the wells ofMillipore, MultiScreenHTS HV filter plates containing 200 μL of water.Liquid was filtered through the plates under vacuum, and 150 μL of HySoymedium was added to each well and filtered through. The filter plateswere then incubated at 37° C., 5% CO₂ overnight and were then fixed withDestain Solution (Bio-Rad). The plates were then stained with CoomassieBlue and destained once. Colonies were imaged and enumerated on aCellular Technology Limited (CTL) ImmunoSpot Analyzer®. The OPA antibodytiter was interpolated from the reciprocal of the two serum dilutionsencompassing the point of 50% reduction in the number of bacterialcolonies when compared to the control wells that did not contain immuneserum.

Particular Embodiments of the Invention are Set Forth in the FollowingNumbered Paragraphs:

1. A glycoconjugate comprising a saccharide covalently conjugated to acarrier protein through a spacer containing ((2-oxoethyl)thio).

2. The glycoconjugate of paragraph 1 having the general formula (I):

wherein:A is a group (C═X)m wherein X is S or O and m is 0 or 1;B is a bond, O, or CH₂; and when m is 0, B can also be (C═O);R is a C₂-C₁₆ alkylene, C₂-C₁₆ heteroakylene, NH—C(═O)—C₂-C₁₆ alkylene,or NH—C(═O)—C₂-C₁₆ heteroakylene, wherein said alkylene andheteroalkylene are optionally substituted by 1, 2 or 3 groupsindependently selected from COOR′ where R′ is selected from H, methyl,ethyl or propyl.

3. The glycoconjugate of paragraph 2 wherein X is O and m is 1.

4. The glycoconjugate of paragraph 2 wherein X is S and m is 1.

5. The glycoconjugate of paragraph 2 wherein m is 0.

6. The glycoconjugate of anyone of paragraphs 2 to 5 wherein B is abond.

7. The glycoconjugate of anyone of paragraphs 2 to 5 wherein B is O.

8. The glycoconjugate of anyone of paragraphs 2 to 5 wherein B is CH₂.

9. The glycoconjugate of paragraph 2 wherein m is 0 and B is (C═O).

10. The glycoconjugate of anyone of paragraphs 2 to 9 wherein R isselected from the groups consisting of (CH₂)₂, (CH₂)₃, (CH₂)₄, (CH₂)₅,(CH₂)₆, (CH₂)₇, (CH₂)₈, (CH₂)₉ or (CH₂)₁₀.

11. The glycoconjugate of anyone of paragraphs 2 to 9 wherein R is aC₂-C₁₆ heteroakylene.

12. The glycoconjugate of anyone of paragraphs 2 to 9 wherein R isselected from the groups consisting of O—CH₂, O—CH₂—CH₂,CH₂—CH₂—O—CH₂—CH₂, O—CH₂—CH₂—O—CH₂—CH₂, O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂,O—CH₂—CH₂—(N—CH₃)—CH₂—CH₂—O—CH₂—CH₂, CH₂—CH₂—(N—CH₃)—CH₂—CH₂ andCH₂—CH₂—S—CH₂—CH₂.

13. The glycoconjugate of anyone of paragraphs 1 to 12 with the provisiothat said spacer is not the (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC)spacer.

14. The glycoconjugate of paragraph 1 having the general formula (II):

wherein R is (CH₂)_(n) where n is 3 to 10.

15. The glycoconjugate of paragraph 14 wherein n is 3.

16. The glycoconjugate of paragraph 14 wherein n is 4.

17. The glycoconjugate of paragraph 14 wherein n is 5.

18. The glycoconjugate of paragraph 14 wherein n is 6.

19. The glycoconjugate of paragraph 1 having the general formula (III):

wherein R is selected from (CH₂CH₂O)_(m)CH₂CH₂, CH(COOH)(CH₂)_(n),NHCO(CH₂)_(n), NHCO(CH₂CH₂O)_(m)CH₂CH₂, OCH₂(CH₂)_(n) orO(CH₂CH₂O)_(m)CH₂CH₂;wherein n is selected from 1 to 10 and m is selected from 1 to 4.

20. The glycoconjugate of paragraph 19 wherein R is CH(COOH)(CH₂)_(n)and wherein n is selected from 1 to 10.

21. The glycoconjugate of paragraph 19 wherein R is NHCO(CH₂)_(n) andwherein n is selected from 1 to 10.

22. The glycoconjugate of paragraph 19 wherein R is OCH₂(CH₂)_(n) andwherein n is selected from 1 to 10.

23. The glycoconjugate of paragraph 19 wherein R is (CH₂CH₂O)_(m)CH₂CH₂and wherein m is selected from 1 to 4.

24. The glycoconjugate of paragraph 19 wherein R isNHCO(CH₂CH₂O)_(m)CH₂CH₂ and wherein m is selected from 1 to 4.

25. The glycoconjugate of paragraph 19 wherein R is O(CH₂CH₂O)_(m)CH₂CH₂and wherein m is selected from 1 to 4.

26. The glycoconjugate of anyone of paragraphs 19 to 22 wherein n is 1to 5.

27. The glycoconjugate of anyone of paragraphs 19 to 22 wherein n is 1to 4.

28. The glycoconjugate of anyone of paragraphs 19 to 22 wherein n is 1to 3.

29. The glycoconjugate of anyone of paragraphs 19 to 22 wherein n is 1or 2.

30. The glycoconjugate of anyone of paragraphs 19 or 23 to 25 wherein mis 1 to 3.

31. The glycoconjugate of anyone of paragraphs 19 or 23 to 25 wherein mis 1 or 2.

32. The glycoconjugate of anyone of paragraphs 19 or 23 to 25 wherein mis 1.

33. The glycoconjugate of paragraph 1 having the general formula (IV):

wherein R is selected from CH₂(CH₂)_(n), (CH₂CH₂O)_(m)CH₂CH₂,CH(COOH)(CH₂)_(n), NHCO(CH₂)_(n), NHCO(CH₂CH₂O)_(m)CH₂CH₂, OCH₂(CH₂)_(n)or O(CH₂CH₂O)_(m)CH₂CH₂;wherein n is selected from 1 to 10 and m is selected from 1 to 4.

34. The glycoconjugate of paragraph 33 wherein R is CH₂(CH₂)_(n) andwherein n is selected from 1 to 10.

35. The glycoconjugate of paragraph 33 wherein R is CH(COOH)(CH₂)_(n)and wherein n is selected from 1 to 10.

36. The glycoconjugate of paragraph 33 wherein R is NHCO(CH₂)_(n) andwherein n is selected from 1 to 10.

37. The glycoconjugate of paragraph 33 wherein R is OCH₂(CH₂)_(n) andwherein n is selected from 1 to 10.

38. The glycoconjugate of paragraph 33 wherein R is (CH₂CH₂O)_(m)CH₂CH₂and wherein m is selected from 1 to 4.

39. The glycoconjugate of paragraph 33 wherein R isNHCO(CH₂CH₂O)_(m)CH₂CH₂ and wherein m is selected from 1 to 4.

40. The glycoconjugate of paragraph 33 wherein R is O(CH₂CH₂O)_(m)CH₂CH₂and wherein m is selected from 1 to 4.

41. The glycoconjugate of anyone of paragraphs 33 to 37 wherein n is 1to 5.

42. The glycoconjugate of anyone of paragraphs 33 to 37 wherein n is 1to 4.

43. The glycoconjugate of anyone of paragraphs 33 to 37 wherein n is 1to 3.

44. The glycoconjugate of anyone of paragraphs 33 to 37 wherein n is 1or 2.

45. The glycoconjugate of anyone of paragraphs 33 or 38 to 40 wherein mis 1 to 3.

46. The glycoconjugate of anyone of paragraphs 33 or 38 to 40 wherein mis 1 or 2.

47. The glycoconjugate of anyone of paragraphs 33 or 38 to 40 wherein mis 1.

48. The glycoconjugate of paragraph 1 having the general formula (V):

wherein R is selected from CH₂(CH₂)₁, (CH₂CH₂O)_(m)CH₂CH₂,CH(COOH)(CH₂)₁, NHCO(CH₂)₁, NHCO(CH₂CH₂O)_(m)CH₂CH₂, OCH₂(CH₂)_(n) orO(CH₂CH₂O)_(m)CH₂CH₂;wherein n is selected from 1 to 10 and m is selected from 1 to 4.

49. The glycoconjugate of paragraph 48 wherein R is CH₂(CH₂)_(n) andwherein n is selected from 1 to 10.

50. The glycoconjugate of paragraph 48 wherein R is CH(COOH)(CH2)_(n)and wherein n is selected from 1 to 10.

51. The glycoconjugate of paragraph 48 wherein R is NHCO(CH2)_(n) andwherein n is selected from 1 to 10.

52. The glycoconjugate of paragraph 48 wherein R is OCH₂(CH₂)n andwherein n is selected from 1 to 10.

53. The glycoconjugate of paragraph 48 wherein R is (CH₂CH₂O)_(m)CH₂CH₂and wherein m is selected from 1 to 4.

54. The glycoconjugate of paragraph 48 wherein R isNHCO(CH₂CH₂O)_(m)CH2CH2 and wherein m is selected from 1 to 4.

55. The glycoconjugate of paragraph 48 wherein R is O(CH₂CH₂O)_(m)CH₂CH₂and wherein m is selected from 1 to 4.

56. The glycoconjugate of anyone of paragraphs 48 to 52 wherein n is 1to 5.

57. The glycoconjugate of anyone of paragraphs 48 to 52 wherein n is 1to 4.

58. The glycoconjugate of anyone of paragraphs 48 to 52 wherein n is 1to 3.

59. The glycoconjugate of anyone of paragraphs 48 to 52 wherein n is 1or 2.

60. The glycoconjugate of anyone of paragraphs 48 or 53 to 55 wherein mis 1 to 3.

61. The glycoconjugate of anyone of paragraphs 48 or 53 to 55 wherein mis 1 or 2.

62. The glycoconjugate of anyone of paragraphs 48 or 53 to 55 wherein mis 1.

63. The glycoconjugate of paragraph 1 comprising a saccharide conjugatedto a carrier protein through an oxo-eTAC spacer, wherein the saccharideis covalently linked to the oxo-eTAC spacer through a carbamate linkage,and wherein the carrier protein is covalently linked to the oxo-eTACspacer through a thioether and amide linkage.

64. The glycoconjugate of paragraph 1 comprising a saccharide conjugatedto a carrier protein through an oxo-eTAAN spacer, wherein the saccharideis covalently linked to the oxo-eTAAN spacer through an amine linkage,and wherein the carrier protein is covalently linked to the oxo-eTAANspacer through a thioether and amide linkage.

65. The glycoconjugate of paragraph 1 comprising a saccharide conjugatedto a carrier protein through an oxo-eTAAD spacer, wherein the saccharideis covalently linked to the oxo-eTAAD spacer through an amide linkage,and wherein the carrier protein is covalently linked to the oxo-eTAADspacer through a thioether and amide linkage.

66. The glycoconjugate of anyone of paragraphs 1 to 65, wherein thesaccharide is a polysaccharide or an oligosacharide.

67. The glycoconjugate of anyone of paragraphs 1 to 65, wherein thesaccharide is a capsular polysaccharide derived from bacteria.

68. The glycoconjugate of paragraph 67, wherein said capsularpolysaccharide is derived from S. pneumoniae.

69. The glycoconjugate of paragraph 68, wherein said capsularpolysaccharide is selected from the group consisting of Pn-serotype 1,2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 17F,18C, 19A, 19F, 20, 22F, 23A, 23B, 23F, 33F and 35B capsularpolysaccharides.

70. The glycoconjugate of paragraph 68, wherein said capsularpolysaccharide is selected from the group consisting of Pn-serotype 2,9N, 15A, 17F, 20, 23A, 23B, and 35B capsular polysaccharides.

71. The glycoconjugate of paragraph 68, wherein said capsularpolysaccharide is selected from the group consisting of Pn-serotype 1,3, 4, 5, 8, 9V, 9N, 12F and 22F.

72. The glycoconjugate of paragraph 68, wherein said capsularpolysaccharide is a Pn-serotype 33F capsular polysaccharide.

73. The glycoconjugate of paragraph 68, wherein said capsularpolysaccharide is a Pn-serotype 22F capsular polysaccharide.

74. The glycoconjugate of paragraph 68, wherein said capsularpolysaccharide is a Pn-serotype 10A capsular polysaccharide.

75. The glycoconjugate of paragraph 68, wherein said capsularpolysaccharide is a Pn-serotype 11A capsular polysaccharide.

76. The glycoconjugate of anyone of paragraphs 1 to 65, wherein thesaccharide is a capsular saccharide derived from N. meningitidis.

77. The glycoconjugate of paragraph 75, wherein the saccharide is apolysaccharide.

78. The glycoconjugate of paragraph 75, wherein the saccharide is anoligosaccharide.

79. The glycoconjugate of paragraphs 75 to 78, wherein the capsularsaccharide is selected from the group consisting of Mn-serotype A, C,W135 and Y capsular saccharides, preferably from the group consisting ofMn-serotype C, W135 and Y capsular saccharides.

80. The glycoconjugate of paragraphs 75 to 78, wherein the capsularsaccharide is of Mn-serotype X capsular saccharide

81. The glycoconjugate of paragraph 67, wherein said capsularpolysaccharide is derived from Group B Streptococcus.

82. The glycoconjugate of paragraph 81, wherein said Group BStreptococcus capsular polysaccharide is selected from the groupconsisting of serotype Ia, Ib, II, III or V capsular polysaccharides

83. The glycoconjugate of paragraph 67, wherein said capsularpolysaccharide is derived from Staphylococcus aureus.

84. The glycoconjugate of paragraph 83, wherein said S. aureus capsularpolysaccharide is S. aureus serotype 5 or 8 capsular polysaccharide.

85. The glycoconjugate of paragraph 67, wherein said capsularpolysaccharide is derived from an Enterococcus bacteria.

86. The glycoconjugate of paragraph 85, wherein said Enterococcuscapsular polysaccharide is Enterococcus faecalis or Enterococcus faeciumcapsular polysaccharide.

87. The glycoconjugate of paragraph 67, wherein said capsularpolysaccharide is derived from a sialic acid and/or uronic acidcontaining bacterial polysaccharide.

88. The glycoconjugate of any one of paragraphs 1 to 87, wherein thesaccharide has a molecular weight of between 10 kDa and 2,000 kDa.

89. The glycoconjugate of paragraph 88, wherein the polysaccharide has amolecular weight of between 50 kDa and 2,000 kDa.

90. The glycoconjugate of any one of paragraphs 1 to 89, wherein theglycoconjugate has a molecular weight of between 50 kDa and 20,000 kDa.

91. The glycoconjugate of paragraph 90, wherein the glycoconjugate has amolecular weight of between 500 kDa and 10,000 kDa.

92. The glycoconjugate of any one of paragraphs 1 to 91, wherein thepolysaccharide has a degree of 0-acetylation between 75-100%.

93. The glycoconjugate of any one of paragraphs 1 to 92, wherein thecarrier protein is selected in the group consisting of TT, DT, DTmutants (such as CRM₁₉₇), H. influenzae protein D, PhtX, PhtD, PhtDEfusions, detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin Aor B of C. Difficile and PsaA.

94. The glycoconjugate of any one of paragraphs 1 to 92, wherein thecarrier protein is selected in the group consisting of TT, DT, CRM₁₉₇and H. influenzae protein D.

95. The glycoconjugate of any one of paragraphs 1 to 92, wherein thecarrier protein is CRM₁₉₇.

96. The glycoconjugate of paragraph 95, wherein the CRM₁₉₇ comprises 2to 20 lysine residues covalently linked to the polysaccharide through anoxo-eT spacer.

97. The glycoconjugate of paragraph 95, wherein the CRM₁₉₇ comprises 4to 16 lysine residues covalently linked to the polysaccharide through anoxo-eT spacer.

98. The glycoconjugate of any one of paragraphs 1 to 97, wherein thesaccharide:carrier protein ratio (w/w) is between 0.2 and 4.

99. The glycoconjugate of paragraph 98, wherein the saccharide:carrierprotein ratio (w/w) is between 0.4 and 1.7.

100. The glycoconjugate of any one of paragraphs 1 to 99, wherein atleast one linkage between the carrier protein and saccharide occurs atevery 25 saccharide repeat units of the saccharide.

101. The glycoconjugate of paragraph 100, wherein at least one linkagebetween the carrier protein and saccharide occurs at every 15 sacchariderepeat units of the saccharide.

102. The glycoconjugate of paragraph 100, wherein at least one linkagebetween the carrier protein and saccharide occurs at every 10 sacchariderepeat units of the saccharide.

103. The glycoconjugate of paragraph 100, wherein at least one linkagebetween the carrier protein and saccharide occurs at every 4 sacchariderepeat units of the saccharide.

104. The glycoconjugate of any one of paragraphs 98 to 103, wherein saidcarrier protein is CRM₁₉₇.

105. The glycoconjugate of any one of paragraphs 1 to 104, whichcomprises less than 15% free saccharide compared to the total amount ofsaccharide.

106. The glycoconjugate of any one of paragraphs 1 to 105, having amolecular size distribution (Kd) of ≧35% at ≦0.3.

107. An immunogenic composition comprising at least one glycoconjugateas defined in anyone of paragraphs 1 to 106.

108. An immunogenic composition comprising at least one glycoconjugateas defined in anyone of paragraphs 1 to 106 and a pharmaceuticallyacceptable excipient, carrier or diluents

109. The immunogenic composition of paragraph 107 or 108, furthercomprising an additional antigen.

110. The immunogenic composition of paragraph 109, wherein theadditional antigen comprises a protein antigen or a glycoconjugate of acapsular polysaccharide derived from S. pneumonia.

111. The immunogenic composition of paragraph 110, wherein theadditional antigen comprises a glycoconjugate of a capsularpolysaccharide selected from the group consisting of Pn-serotype 1, 3,4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23Fand 33F capsular polysaccharides.

112. The immunogenic composition of paragraph 109, wherein theadditional antigen comprises a protein antigen or a glycoconjugate of acapsular polysaccharide derived from N. meningitidis.

113. The immunogenic composition of paragraph 109, wherein theadditional antigen comprises a glycoconjugate of a capsularpolysaccharide selected from the group consisting of serotype A, C, W135and Y capsular polysaccharides.

114. The immunogenic composition of any one of paragraphs 107 to 113,further comprising an adjuvant.

115. The immunogenic composition of paragraph 114, wherein the adjuvantis an aluminum-based adjuvant.

116. The immunogenic composition of paragraph 114, wherein the adjuvantis an aluminum phosphate.

117. The immunogenic composition of paragraph 114, wherein the adjuvantis an aluminum hydroxide.

118. A container filled with any of the immunogenic composition definedat any of one of paragraphs 107 to 117.

119. The container of paragraph 118 selected from the group consistingof a vial, a syringe, a flask, a fermentor, a bioreactor, a bag, a jar,an ampoule, a cartridge and a disposable pen.

120. A method of making a glycoconjugate comprising a saccharideconjugated to a carrier protein through a(((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC) spacer, comprising thesteps of:

-   -   a) reacting a saccharide with a carbonic acid derivative or        cyanogen derivative, to produce an activated saccharide;    -   b) reacting the activated saccharide with a bifunctional linker        containing amine and thiol functionalities or a salt thereof, to        produce a thiolated saccharide;    -   c) reacting the thiolated saccharide with a deprotecting or        reducing agent to produce an activated thiolated saccharide        comprising one or more free sulfhydryl residues;    -   d) reacting the activated thiolated saccharide with an activated        carrier protein comprising one or more α-haloacetamide groups,        to produce a thiolated saccharide-carrier protein conjugate; and    -   e) reacting the thiolated saccharide-carrier protein conjugate        with (i) a first capping reagent capable of capping unconjugated        α-haloacetamide groups of the activated carrier protein;        and/or (ii) a second capping reagent capable of capping        unconjugated free sulfhydryl residues of the activated thiolated        saccharide;    -   whereby an oxo-eTAC linked glycoconjugate is produced

121. The method of paragraph 120, wherein the carbonic acid derivativeof step a) is 1,1′-carbonyl-di-(1,2,4-triazole) (CDT) or1,1′-carbonyldiimidazole (CDI) or disuccinimidyl carbonate (DSC) orN-hydroxysuccinimidyl chloroformate.

122. The method of paragraph 121, wherein the carbonic acid derivativeof step a) is 1,1′-carbonyl-di-(1,2,4-triazole) (CDT).

123. The method of anyone of paragraphs 120 to 122, wherein step a) isdone in an organic solvent.

124. The method of paragraph 123, wherein said organic solvent is apolar aprotic solvent, such as dimethylsulfoxide (DMSO).

125. The method of anyone of paragraphs 120 to 124, wherein thethiolated saccharide is produced by reaction of the activated saccharidewith a heterobifunctional thioalkylamine reagent or a salt thereof.

126. The method of anyone of paragraphs 120 to 125, wherein the firstcapping reagent is N-acetyl-L-cysteine.

127. The method of anyone of paragraphs 120 to 126, wherein the secondcapping reagent is iodoacetamide (IAA).

128. The method of anyone of paragraphs 120 to 127, wherein step e)comprises capping with both a first capping reagent and a second cappingreagent.

129. The method of anyone of paragraphs 120 to 125, wherein step e)comprises capping with N-acetyl-L-cysteine as the first capping reagentand IAA as the second capping reagent.

130. The method of anyone of paragraphs 120 to 129, wherein the cappingstep e) further comprises reaction with a reducing agent, for example,DTT, TCEP, or mercaptoethanol, after reaction with the first and/orsecond capping reagent.

131. The method of anyone of paragraphs 120 to 129, wherein step d)further comprises providing an activated carrier protein comprising oneor more α-haloacetamide groups prior to reacting the activated thiolatedsaccharide with the activated carrier protein.

132. The method of anyone of paragraphs 131, wherein the activatedcarrier protein comprises one or more α-bromoacetamide groups.

133. The method of anyone of paragraphs 120 to 132, wherein saidactivated carrier protein is activated CRM197 carrier protein comprisingone or more α-bromoacetamide groups.

134. A method of making a glycoconjugate comprising a saccharideconjugated to a carrier protein through an(((2-oxoethyl)thio)alkyl)amine (oxo-eTAAN) spacer, comprising the stepsof:

-   -   a) reacting a saccharide with an oxidizing reagent to generate        aldehyde groups to produce an activated saccharide;    -   b) reacting the activated saccharide with a bifunctional linker        containing amine and thiol functionalities (in protected or free        forms) from the amino end of the linker, to produce a thiolated        saccharide by reductive amination;    -   c) reacting the thiolated saccharide with a deprotecting agent        or reducing agent (if thiol is protected) to produce an        activated thiolated saccharide comprising one or more free        sulfhydryl residues;    -   d) reacting the activated thiolated saccharide with an activated        carrier protein comprising one or more α-haloacetamide groups,        to produce a thiolated saccharide-carrier protein conjugate; and    -   e) reacting the thiolated saccharide-carrier protein conjugate        with (i) a first capping reagent capable of capping unconjugated        α-haloacetamide groups of the activated carrier protein;        and/or (ii) a second capping reagent capable of capping        unconjugated free sulfhydryl residues of the activated thiolated        saccharide;    -   whereby an oxo-eTAAN linked glycoconjugate is produced.

135. The method of paragraph 134 wherein the saccharide of step a) isoxidized by 2,2,6,6-Tetramethyl-1-piperidinyloxy(TEMPO)/N-Chlorosuccinimide (NCS) reagent system.

136. A method of making a comprising a carboxyl containing saccharideconjugated to a carrier protein through an(((2-oxoethyl)thio)alkyl)amide (oxo-eTAAD) spacer, comprising the stepsof:

-   -   a) first reacting the carboxyl group containing saccharide to        generated an activated saccharide with a carbodiimide or a        derivative thereof;    -   b) reacting the activated saccharide with a heterobifunctional        linker containing amine and thiol functionalities (in protected        or free form) from the amino end, to produce a thiolated        saccharide;    -   c) reacting the thiolated saccharide with a deprotecting or        reducing agent (if protected) to produce an activated thiolated        saccharide comprising one or more free sulfhydryl residues;    -   d) reacting the activated thiolated saccharide with an activated        carrier protein comprising one or more α-haloacetamide groups,        to produce a thiolated saccharide-carrier protein conjugate; and    -   e) reacting the thiolated saccharide-carrier protein conjugate        with (i) a first capping reagent capable of capping unconjugated        α-haloacetamide groups of the activated carrier protein;        and/or (ii) a second capping reagent capable of capping        unconjugated free sulfhydryl residues of the activated thiolated        saccharide;    -   whereby an oxo-eTAAD linked glycoconjugate is produced.

137. The method of paragraph 136 wherein the carbodiimide derivative is1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), orN,N′-dicyclohexylcarbodiimide (DCC), or1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide.

138. The method of paragraph 136 wherein the carbodiimide derivative is1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

139. The method of anyone of paragraphs 136 to 138, wherein step a) isdone in an organic solvent.

140. The method of paragraph 139, wherein said organic solvent is apolar aprotic solvent, such as dimethylsulfoxide (DMSO).

141. The method of anyone of paragraphs 136 to 140 wherein thecarboxylic acid activation step a) is accomplished by carbodiimide andthiazolidinone thione.

142. The method of anyone of paragraphs 136 to 140 wherein thecarboxylic acid activation step a) is accomplished byN-ethyl-3-phenylisoxazolium-3′-sulfonate (Woodward's reagent K).

143. The method of anyone of paragraphs 120 to 142, further comprisingpurification of the thiolated polysaccharide produced in step c),wherein the purification step comprises diafiltration.

144. The method of any one of paragraphs 120 to 143, wherein the carrierprotein is activated with an activated bromoacetic acid derivative.

145. The method of paragraph 144, wherein the bromoacetic acidderivative is the N-hydroxysuccinimide ester of bromoacetic acid(BAANS).

146. The method of any one of paragraphs 120 to 145, wherein the methodfurther comprises purification of the glycoconjugate by diafiltration.

147. The method of any one of paragraphs 120 to 146, wherein step a) isconducted in a polar aprotic solvent selected from the group consistingof dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide(DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile,1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) andhexamethylphosphoramide (HMPA), or a mixture thereof.

148. The method of any one of paragraphs 120 to 147, wherein thesaccharide:carrier protein ratio (w/w) is between 0.2 and 4.

149. The method of paragraph 148, wherein the saccharide:carrier proteinratio (w/w) is between 0.4 and 1.7.

150. The method of any one of paragraphs 120 to 149, wherein thesaccharide is a capsular polysaccharide derived from S. pneumoniae.

151. The method of paragraph 150, wherein the capsular polysaccharide isselected from the group consisting of pneumococcal (Pn) serotype 1, 3,4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23Fand 33F capsular polysaccharides.

152. The method of paragraph 150, wherein the capsular polysaccharide isa Pn-serotype 33F capsular polysaccharide.

153. The method of paragraph 150, wherein the capsular polysaccharide isa Pn-serotype 22F capsular polysaccharide.

154. The method of paragraph 150, wherein the capsular polysaccharide isa Pn-serotype 10A capsular polysaccharide.

155. The method of paragraph 150, wherein the capsular polysaccharide isa Pn-serotype 11A capsular polysaccharide.

156. The method of any one of paragraphs 120 to 149, wherein thesaccharide is a capsular polysaccharide derived from N. meningitidis.

157. The method of any one of paragraphs 120 to 156, wherein the carrierprotein is CRM₁₉₇.

158. A glycoconjugate produced by the method of any one of paragraphs120 to 157.

159. An immunogenic composition comprising the glycoconjugate ofparagraph 158 and a pharmaceutically acceptable excipient, carrier ordiluent.

160. The immunogenic composition of paragraph 159 further comprising anadjuvant.

161. The immunogenic composition of paragraph 160, wherein thealuminum-based adjuvant is selected from the group consisting ofaluminum phosphate, aluminum sulfate, and aluminum hydroxide

162. A method of preventing, treating or ameliorating a bacterialinfection, disease or condition in a subject, comprising administeringto the subject an immunologically effective amount of an immunogeniccomposition of any one of paragraphs 107 to 117 or 159 to 161.

163. A method of inducing a protective immune response in a subject,comprising administering to the subject an immunologically effectiveamount of an immunogenic composition of any one of paragraphs 107 to 117or 159 to 161.

164. The glycoconjugates of anyone of paragraph 1 to 106 or theimmunogenic composition of any one of paragraphs 107 to 117 or 159 to161 for use as a medicament.

165. The glycoconjugates of anyone of paragraph 1 to 106 or theimmunogenic composition of any one of paragraphs 107 to 117 or 159 to161 for use as a vaccine.

166. The glycoconjugates of anyone of paragraph 1 to 106 or theimmunogenic composition of any one of paragraphs 107 to 117 or 159 to161 for use in a method of preventing, treating or ameliorating abacterial infection, disease or condition in a subject.

167. The glycoconjugates of anyone of paragraph 1 to 106 or theimmunogenic composition of any one of paragraphs 107 to 117 or 159 to161 for use in a method of preventing, a bacterial infection in asubject.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theinvention.

EXAMPLES Example 1 General Process for Preparation of eTEC LinkedGlycoconjugates Activation of Saccharide and Thiolation with CystamineDihydrochloride

The saccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO).Moisture content of the solution is determined by Karl Fischer (KF)analysis and adjusted to reach a moisture content of 0.1 and 0.4%,typically 0.2%.

To initiate the activation, a solution of1,1′-carbonyl-di-1,2,4-triazole (CDT) or 1,1′-carbonyldiimidazole (CDI)is freshly prepared at a concentration of 100 mg/mL in DMSO. Thesaccharide is activated with various amounts of CDT/CDI (1-10 molarequivalents) and the reaction is allowed to proceed for 1 hour at 23±2°C. The activation level may be determined by HPLC. Cystaminedihydrochloride is freshly prepared in anhydrous DMSO at a concentrationof 50 mg/mL. The activated saccharide is reacted with 1 mol. eq. ofcystamine dihydrochloride. Alternatively, the activated saccharide isreacted with 1 mol. eq. of cysteamine hydrochloride. The thiolationreaction is allowed to proceed for 21±2 hours at 23±2° C., to produce athiolated saccharide. The thiolation level is determined by the addedamount of CDT/CDI.

Residual CDT/CDI in the activation reaction solution is quenched by theaddition of 100 mM sodium tetraborate, pH 9.0 solution. Calculations areperformed to determine the added amount of tetraborate and to adjust thefinal moisture content to be up to 1-2% of total aqueous.

Reduction and Purification of Activated Thiolated Saccharide

The thiolated saccharide reaction mixture is diluted 10-fold by additionto pre-chilled 5 mM sodium succinate in 0.9% saline, pH 6.0 and filteredthrough a 5 μm filter. Diafiltration of thiolated saccharide isperformed against 40-fold diavolume of WFI. To the retentate a solutionof tris(2-carboxyethyl)phosphine (TCEP), 1-5 mol. eq., is added afterdilution by 10% volume of 0.1M sodium phosphate buffer, pH 6.0. Thisreduction reaction is allowed to proceed for 20±2 hours at 5±3° C.Purification of the activated thiolated saccharide is performedpreferably by ultrafiltration/dialfiltration of against pre-chilled 10mM sodium phosphate monobasic, pH 4.3. Alternatively, the thiolatedsaccharide is purified by standard size exclusion chromatographic (SEC)procedures or ion exchange chromatographic methods. An aliquot ofactivated thiolated saccharide retentate is pulled to determine thesaccharide concentration and thiol content (Ellman) assays.

Alternative Reduction and Purification of Activated Thiolated Saccharide

As an alternative to the purification procedure described above,activated thiolated saccharide was also purified as below.

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 5-10 mol. eq., was added andallowed to proceed for 3±1 hours at 23±2° C. The reaction mixture wasthen diluted 5-fold by addition to pre-chilled 5 mM sodium succinate in0.9% saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltrationof thiolated saccharide was performed using 40-fold diavolume ofpre-chilled 10 mM sodium phosphate monobasic, pH 4.3. An aliquot ofactivated thiolated saccharide retentate was pulled to determine thesaccharide concentration and thiol content (Ellman) assays.

Activation and Purification of Bromoacetylated Carrier Protein

Free amino groups of the carrier protein are bromoacteylated by reactionwith a bromoacetylating agent, such as bromoacetic acidN-hydroxysuccinimide ester (BAANS), bromoacetylbromide, or anothersuitable reagent.

The carrier protein (in 0.1M Sodium Phosphate, pH 8.0±0.2) is first keptat 8±3° C., at about pH 7 prior to activation. To the protein solution,the N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a stockdimethylsulfoxide (DMSO) solution (20 mg/mL) is added in a ratio of0.25-0.5 BAANS: protein (w/w). The reaction is gently mixed at 5±3° C.for 30-60 minutes. The resulting bromoacetylated (activated) protein ispurified, e.g., by ultrafiltration/diafiltration using 10 kDa MWCOmembrane using 10 mM phosphate (pH 7.0) buffer. Following purification,the protein concentration of the bromoacetylated carrier protein isestimated by Lowry protein assay.

The extent of activation is determined by total bromide assay byion-exchange liquid chromatography coupled with suppressed conductivitydetection (ion chromatography). The bound bromide on the activatedbromoacetylated protein is cleaved from the protein in the assay samplepreparation and quantitated along with any free bromide that may bepresent. Any remaining covalently bound bromine on the protein isreleased by conversion to ionic bromide by heating the sample inalkaline 2-mercaptoethanol.

Activation and Purification of Bromoacetylated CRM₁₉₇

CRM₁₉₇ was diluted to 5 mg/mL with 10 mM phosphate buffered 0.9% NaCl pH7 (PBS) and then made 0.1 M NaHCO₃ pH 7.0 using 1 M stock solution.BAANS was added at a CRM₁₉₇: BAANS ratio 1:0.35 (w:w) using a BAANSstock solution of 20 mg/mL DMSO. The reaction mixture was incubated atbetween 3° C. and 11° C. for 30 mins-1 hour then purified byultrafiltration/diafiltration using a 10K MWCO membrane and 10 mM SodiumPhosphate/0.9% NaCl, pH 7.0. The purified activated CRM₁₉₇ was assayedby the Lowry assay to determine the protein concentration and thendiluted with PBS to 5 mg/mL. Sucrose was added to 5% wt/vol as acryoprotectant and the activated protein was frozen and stored at −25°C. until needed for conjugation.

Bromoacetylation of lysine residues of CRM₁₉₇ was very consistent,resulting in the activation of 15 to 25 lysines from 39 lysinesavailable. The reaction produced high yields of activated protein.

Conjugation of Activated Thiolated Saccharide to Bromoacetylated CarrierProtein

Before starting the conjugation reaction, the reaction vessels arepre-cooled to 5° C. Bromoacetylated carrier protein and activatedthiolated saccharide are subsequently added and mixed at an agitationspeed of 150-200 rpm. The saccharide/protein input ratio is 0.9±0.1. Thereaction pH is adjusted to 8.0±0.1 with 1 M NaOH solution. Theconjugation reaction is allowed to proceed at 5° C. for 20±2 hours.

Capping of Residual Reactive Functional Groups

The unreacted bromoacetylated residues on the carrier protein arequenched by reacting with 2 mol. eq. of N-acetyl-L-cysteine as a cappingreagent for 3 hours at 5° C. Residual free sulfhydryl groups are cappedwith 4 mol. eq. of iodoacetamide (IAA) for 20 hours at 5° C.

Purification of eTEC-Linked Glycoconjugate

The conjugation reaction (post-IAA-capped) mixture is filtered through0.45 μm filter. Ultrafiltration/dialfiltration of the glycoconjugate isperformed against 5 mM succinate-0.9% saline, pH 6.0. The glycoconjugateretentate is then filtered through 0.2 μm filter. An aliquot ofglycoconjugate is pulled for assays. The remaining glycoconjugate isstored at 5° C.

Example 2 Preparation of Pn-33F eTEC Conjugates Activation Process

Activation of Pn33F Polysaccharide

Pn-33F polysaccharide was compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture was shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized 33F polysaccharide was reconstituted inanhydrous dimethylsulfoxide (DMSO). Moisture content of the lyophilized33F/DMSO solution was determined by Karl Fischer (KF) analysis. Themoisture content was adjusted by adding WFI to the 33F/DMSO solution toreach a moisture content of 0.2%.

To initiate the activation, 1,1′-carbonyl-di-1,2,4-triazole (CDT) wasfreshly prepared as 100 mg/mL in DMSO solution. Pn33F polysaccharide wasactivated with various amounts of CDT prior to the thiolation step. TheCDT activation was carried out at 23±2° C. for 1 hour. The activationlevel was determined by HPLC (A220/A205). Sodium tetraborate, 100 mM, pH9.0 solution was added to quench any residual CDT in the activationreaction solution. Calculations are performed to determine the addedamount of tetraborate and to allow the final moisture content to be 1.2%of total aqueous. The reaction was allowed to proceed for 1 hour at23±2° C.

Thiolation of Activated Pn-33F Polysaccharide

Cystamine-dihydrochloride was freshly prepared in anhydrous DMSO and 1mol. eq. of cystamine dihydrochloride was added to the activatedpolysaccharide reaction solution. The reaction was allowed to proceedfor 21±2 hours at 23±2° C. The thiolated saccharide solution was diluted10-fold by addition to pre-chilled 5 mM sodium succinate in 0.9% saline,pH 6.0. The diluted reaction solution was filtered through a 5 μmfilter. Dialfiltration of thiolated Pn-33F polysaccharide was carriedout with 100K MWCO ultrafilter membrane cassettes, using Water forInjection (WFI).

Reduction and Purification of Activated Thiolated Pn-33F Polysaccharide

To the retentate a solution of tris(2-carboxyethyl)phosphine (TCEP), 5mol. eq., was added after dilution by 10% volume of 0.1M sodiumphosphate buffer, pH 6.0. This reduction reaction was allowed to proceedfor 2±1 hours at 23±2° C. Dialfiltration of thiolated 33F polysaccharidewas carried out with 100K MWCO ultrafilter membrane cassettes.Diafiltration was performed against pre-chilled 10 mM sodium phosphate,pH 4.3. The thiolated 33F polysaccharide retentate was pulled for bothsaccharide concentration and thiol (Ellman) assays.

Alternative Reduction and Purification of Activated Thiolated Pn-33FPolysaccharide

As an alternative to the purification procedure described above, 33Factivated thiolated saccharide was also purified as follows.

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 5 mol. eq., was added and allowedto proceed for 3±1 hours at 23±2° C. The reaction mixture was thendiluted 5-fold by addition to pre-chilled 5 mM sodium succinate in 0.9%saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltration ofthiolated saccharide was performed using 40-fold diavolume ofpre-chilled 10 mM sodium phosphate monobasic, pH 4.3 with 100K MWCOultrafilter membrane cassettes. The thiolated 33F polysaccharideretentate was pulled for both saccharide concentration and thiol(Ellman) assays.

Conjugation Process

Conjugation of Thiolated Pn33F Polysaccharide to Bromoacetylated CRM₁₉₇

The CRM₁₉₇ carrier protein was activated separately by bromoacetylation,as described in Example 1, and then reacted with the activated Pn-33Fpolysaccharide for the conjugation reaction. Before starting theconjugation reaction, the reaction vessel was pre-cooled to 5° C.Bromoacetylated CRM₁₉₇ and thiolated 33F polysaccharide were mixedtogether in a reaction vessel at an agitation speed of 150-200 rpm. Thesaccharide/protein input ratio was 0.9±0.1. The reaction pH was adjustedto 8.0-9.0. The conjugation reaction was allowed to proceed at 5° C. for20±2 hours.

Capping of Reactive Groups on Bromoacetylated CRM₁₉₇ and Thiolated Pn33FPolysaccharide

The unreacted bromoacetylated residues on CRM₁₉₇ proteins were capped byreacting with 2 mol. eq. of N-acetyl-L-cysteine for 3 hours at 5° C.,followed by capping any residual free sulfhydryl groups of the thiolated33F-polysaccharide with 4 mol. eq. of iodoacetamide (IAA) for 20 hoursat 5° C.

Purification of eTEC-Linked Pn-33F Glycoconjugate

The conjugation solution was filtered through a 0.45 μm or 5 μm filter.Dialfiltration of the 33F glycoconjugate was carried out with 300K MWCOultrafilter membrane cassettes. Diafiltration was performed against 5 mMsuccinate-0.9% saline, pH 6.0. The Pn-33F glycoconjugate 300K retentatewas then filtered through a 0.22 μm filter and stored at 5° C.

Results

The reaction parameters and characterization data for several batches ofPn-33F eTEC glycoconjugates are shown in Table 2. The CDTactivation-thiolation with cystamine dihydrochloride generatedglycoconjugates having from 63 to 90% saccharide yields and <1% to 13%free saccharides.

TABLE 2 Experimental Parameters and Characterization Data of Pn33F eTECConjugates Conjugate Batch 33F-1A 33F-2B 33F-3C 33F-4D 33F-5E 33F-6F33F-7G Activation level (mol of 0.21 0.13  0.164  0.103  0.183 0.22 0.19thiol/mol of polysaccharide) Activation level (% Thiol) 21    13   16.4  10.3  18.3  22 19 Saccharide/Protein 0.75 1.0  0.75 1.0 1.0 0.750.80 (Input) ratio Saccharide yield (%)   69% 63% 71% 63% 69% 82% 90%Saccharide/Protein Ratio 1.3  1.7  1.2  1.9 1.6 1.1 1.5 Free Saccharide12.9% 7.7%  4.4%  7.2%  7.3%  <4% <4% MW by SEC-MALLS (kDa) 2627    2561     4351     2981    3227    3719 5527 CMCA/CMC 14.4/0 13.4/06.8/1.9 2.7/0.6 5.9/0.6 8.2/0 11.4/0.6 % Kd (≦0.3) N/A 85% 88% 75% 68%67% 76% Acetylation level (mol of 0.89 1.16 0.99  0.85  0.81 0.85 1.01acetate/mol of polysaccharide)

OPA Titers of Pn-33F eTEC Glycoconjugates to CRM₁₉₇

Pn-33F OPA titers in mice were determined under standard conditions. OPAtiters (GMT with 95% CI) at four and seven weeks are shown in Table 3,demonstrating that the serotype 33F Pn glycoconjugate elicited OPAtiters in a murine immunogenicity model.

TABLE 3 Pn-33F OPA Titers (GMT with 95% CI) 33F Pn Conjugate 0.001 μg0.01 μg 0.1 μg week 4 4 (4, 5)  37 (17, 82) 414 (234, 734)  week 7 8 (5,13) 131 (54, 314) 17567 (9469, 32593)

Example 3 Preparation of Pn-22F eTEC Conjugates Activation Process

Activation of Pn-22F Polysaccharide

Pn-22F polysaccharide was compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture was shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized 22F polysaccharide was reconstituted inanhydrous dimethylsulfoxide (DMSO). Moisture content of the lyophilized22F/DMSO solution was determined by Karl Fischer (KF) analysis. Themoisture content was adjusted by adding WFI to the Pn-22F/DMSO solutionto reach a moisture content of 0.2%.

To initiate the activation, 1,1′-carbonyl-di-1,2,4-triazole (CDT) wasfreshly prepared as 100 mg/mL in DMSO solution. Pn-22F polysaccharidewas activated with various amounts of CDT followed by thiolation with 1mol. eq. of cystamine dihydrochloride. The CDT activation was carriedout at 23±2° C. for 1 hour. The activation level was determined by HPLC(A220/A205). Sodium tetraborate, 100 mM, pH 9.0 solution was added toquench any residual CDT in the activation reaction solution.Calculations are performed to determine the added amount of tetraborateand to allow the final moisture content to be 1.2% of total aqueous. Thereaction was allowed to proceed for 1 hour at 23±2° C.

Thiolation of Activated Pn-22F Polysaccharide

Cystamine-dihydrochloride was freshly prepared in anhydrous DMSO andadded to the reaction solution. The reaction was allowed to proceed for21±2 hours at 23±2° C. The thiolated saccharide solution was diluted10-fold by addition to pre-chilled 5 mM sodium succinate in 0.9% saline,pH 6.0. The diluted reaction solution was filtered through a 5 μmfilter. Dialfiltration of thiolated Pn-22F polysaccharide was carriedout with 100K MWCO ultrafilter membrane cassettes, using Water forInjection (WFI).

Reduction and Purification of Activated Thiolated Pn-22F Polysaccharide

To the retentate a solution of tris(2-carboxyethyl)phosphine (TCEP),5-10 mol. eq., was added after dilution by 10% volume of 0.1 M sodiumphosphate buffer, pH 6.0. This reduction reaction was allowed to proceedfor 2±1 hours at 23±2° C. Diafiltration of thiolated 22F polysaccharidewas carried out with 100K MWCO ultrafilter membrane cassettes.Diafiltration was performed against pre-chilled 10 mM sodium phosphate,pH 4.3. The thiolated 22F polysaccharide retentate was pulled for bothsaccharide concentration and thiol (Ellman) assays.

Conjugation, Capping and Purification of Pn-22F eTEC Glycoconjugates

Conjugation of the activated thiolated Pn22F polysaccharide to activatedCRM₁₉₇, capping, and purification of the Pn-22F eTEC glycoconjugateswere performed according to the processes described in Example 2.

Results

Characterization and process data for representative Pn-22F eTECglycoconjugates to CRM₁₉₇ is provided in Table 4.

TABLE 4 Experimental Parameters and Characterization Data for Pn-22FeTEC conjugates Conjugate Batch Pn-22F-1A Pn-22F-1B Pn-22F-1C Pn-22F-1DPolysaccharide MW (kDa) 638.5 kDa  638.5 kDa 638.5 kDa  638.5 kDa  PolyActivation Mol. Eq. of CDT 0.6 mol. equiv. 0.9 mol. equiv. 1.2 mol.equiv. 1.5 mol. equiv. Mol. Eq. of Thiol 1 mol. eq of 1 mol. eq of 1mol. eq of 1 mol. eq of cystamine•2HCl cystamine•2HCl cystamine•2HClcystamine•2HCl Mol. Eq. of Reductant 10 mol. eq. of 10 mol. eq. of 10mol. eq. of 10 mol. eq. of TCEP TCEP TCEP TCEP Yield 86% 89% 71% 86%Thiol (Activation) level (mol of 0.05 0.09 0.12 0.16 thiol/mol ofpolysaccharide) Activation level 5 9 12 16 (% Thiol) Conjugation toCRM197 Input Ratio 0.75 0.75 0.75 0.75 Conjugation Results SaccharideYield (%) 55% 48% 56% 35% Saccharide/Protein 1.4 1.2 1.1 1.1 Ratio FreeSaccharide 29.7%   16.8%   9.1%  10.1%   Free Protein <1% <1% <1% <1% Mwby SEC-MALLS 1808 kDa 1787 kDa 1873 kDa 2248 kDa

Example 4 Preparation of Pn-10A eTEC Conjugates to CRM₁₉₇

Preparation of Pn-10A eTEC Glycoconjugates

Glycoconjugates comprising pneumococcal capsular polysaccharide serotype10A (Pn-10A) conjugated to CRM₁₉₇ via the eTEC spacer were preparedaccording to the processes described in Example 2.

Characterization of Pn-10A eTEC Glycoconjugates

Characterization and process data for representative Pn-10A eTECglycoconjugates to CRM₁₉₇ is provided in Table 5.

TABLE 5 Experimental Parameters and Characterization Data for Pn-10AGlycoconjugates Conjugation Batch Pn- Pn- Pn- 10A-1 Pn-10A-2 10A-3Pn-10A-4 10A-5 Saccharide MW 538 128 128 128 128 (kDa) Activation level0.13 0.18 0.29 0.34 0.43 (mol of thiol/mol of polysaccharide) Activationlevel 13 18 29 34 43 (% Thiol) Conjugate MW 2510 950 800 909 1090 (kDa)% Yield 67% 42% 53% 55% 50% (saccharide) % Free Saccharide 20 4.5 <4 <4<4 Kd (% ≦ 0.3) 71% 36% 38% 35% 37% Free Protein <1% <1% <1% <1% <1%CMCA residues N/A 9.8 14.6 15.9 18.5

Pn-10A OPA Titers

OPA titers against the Pn-10A eTEC conjugate to CRM₁₉₇ in mice weredetermined under standard conditions. OPA titers as a function of doseare shown in Table 6. The OPA titers were significantly higher for theconjugate in relation to the unconjugated Serotype 10A polysaccharide.

TABLE 6 Pn-10A OPA Titers (GMT with 95% CI) 10A Pn Variant 0.001 μg 0.01μg 0.1 μg Pn-10A eTEC 691 (389, 1227) 1208 3054 (1897, 4918) conjugate(657, 2220) Unconjugated 602 (193, 1882) PS

Example 5 Preparation of Pn-11A eTEC Conjugates to CRM₁₉₇

Preparation of Pn-11A eTEC Glycoconjugates

Glycoconjugates comprising pneumococcal capsular polysaccharide serotype11A (Pn-11A) conjugated to CRM₁₉₇ via the eTEC spacer were preparedaccording to the processes described in Example 2.

Characterization of Pn-11A eTEC Glycoconjugates

Characterization and process data for representative Pn-11A eTECglycoconjugates to CRM₁₉₇ is provided in Table 7.

TABLE 7 Experimental Parameters and Characterization Data for Pn-11AGlycoconjugates Conjugation Batch Pn- Pn- Pn- Pn- 11A-1A 11A-1B 11A-2A11A-2B Polysaccharide MW 113 kDa 113 kDa 230 kDa 230 kDa Mol. Eq of CDT5 5 2 2 Mol. Eq of Thiol 0.25 0.07 1 1 Mol. Eq of TCEP 10 10 10 10 Yield62% 51% 86% 82% Activation Level (mol of 0.46 0.14 0.13 0.10 thiol/molof polysaccharide) Activation level 46 14 13 10 (% Thiol) Conjugation toCRM₁₉₇ Saccharide/Protein Input 0.75 0.75 0.75 0.75 Ratio SaccharideYield 46.4%   60.4%   73.3%   73.9%   Saccharide/Protein Ratio 0.96 1.91.18 1.23 Free Saccharide <4% 55% 16% 23% Free Protein <1% <1% <1% <1%MW by SEC-MALLS 1203 1074 1524 1884 (kDa)

Pn-11A OPA Titers

OPA titers against the Pn-11A eTEC conjugate to CRM₁₉₇ in mice weredetermined under standard conditions. OPA titers as a function of doseare shown in Table 8.

TABLE 8 Pn-11A OPA Titers (GMT with 95% CI) 11A Pn Variant 0.001 μg 0.01μg 0.1 μg Pn-11A eTEC 206 (166, 256) 906 (624, 1316) 5019 (3648, 6904)conjugate

Example 6 Preparation of Pn-33F RAC/Aqueous Conjugates to CRM₁₉₇

Preparation of Pn-33F RAC/Aqueous Glycoconjugates

Pn-33F glycoconjugates were prepared using Reductive Amination inAqueous Phase (RAC/Aqueous), which has been successfully applied toproduce pneumococcal conjugate vaccine (see e.g. WO 2006/110381). Thisapproach includes two steps. The first step is oxidation ofpolysaccharide to generate aldehyde functionality from vicinal diols.The second step is to conjugate activated polysaccharide to the lysine(Lys) residues of CRM₁₉₇. Briefly, frozen polysaccharide was thawed andoxidation was carried out in sodium phosphate buffer at pH 6.0 by theaddition of different amount of sodium periodate (NaIO4). Concentrationand diafiltration of the activated polysaccharide was carried out andthe purified activated polysaccharide was stored at 4° C. Activatedpolysaccharide was compounded with CRM₁₉₇ protein. Thoroughly mixingpolysaccharide and CRM₁₉₇ is conducted before placing the bottle in dryice/ethanol bath, followed by lyophilization of thepolysaccharide/CRM₁₉₇ mixture. The lyophilized mixture was reconstitutedin 0.1 M sodium phosphate buffer. Conjugation reaction was initiated bythe addition of 1.5 molar equivalents of sodium cyanoborohydride andincubation for 20 hrs at 23° C. and additional 44 hrs at 37° C. Thereactions were diluted with 1× volume of 0.9% saline and capped using 2MEq of sodium borohydride for 3 hrs at 23° C. The reaction mixture wasdiluted with 1× volume of 0.9% saline and then filtered through 0.45 μmfilter prior to purification. Concentration and diafiltration of theconjugate was carried out using 100K MWCO UF membrane cassettes.

Several conjugates were obtained using the above described process byvarying different parameters (e.g. pH, temperature of the reactions andconcentration of polysaccharide).

The typical polysaccharide yield was approximately 50% for theseconjugates and 15% of free saccharide with conjugate MW in the range2000-3500 kDa.

However, native serotype 33F polysaccharide bears an O-Acetyl group onits C2 of 5-galactofuranosyl residue and it was found that ˜80% of theacetyl functional group is removed throughout conjugation process usingReductive Amination in Aqueous Phase. It was observed that the O-Acetylgroup on the five member ring structure (5-galactofuranoside) canmigrate and be removed with ease using Reductive Amination Chemistry inAqueous Phase process.

Evaluation of Pn-33F RAC/Aqueous Glycoconjugate Stability

Aliquots of representative RAC/Aqueous conjugate prepared by the aboveprocess were dispensed into polypropylene tubes. These tubes were storedeither at 25° C. or at 37° C. and stability was monitored up to 3.5months. At each stability time point, % free saccharide levels wereevaluated. The stability data at both temperatures are summarized inTable 9. As shown in Table 9, the % free saccharide levels increasedsignificantly at 25° C. and 37° C. Increase in % free saccharide levelsduring storage is a potential indicator for polysaccharide degradationin the conjugate.

TABLE 9 Stability Data for RAC/Aqueous Conjugate at 25° C. and 37° C.Time Lot# 0 2 wks 1 M 3.5 Ms Free Saccharide (%) at 25° C. 1-B 8.5 14 1420 Free Saccharide (%) at 37° C. 1-B 8.5 17 21 38 wk = week; M = month.

Although, serotype 33F polysaccharide was successfully activated by thereaction with sodium periodate and subsequently conjugated to CRM₁₉₇exploiting aqueous reductive amination chemistry, the % free saccharidestability results under accelerated conditions combined with theinability to preserve the acetyl functionality (a key polysaccharideepitope for immunogenicity) during conjugation suggested that theRAC/aqueous process is not the optimal process for serotype 33Fconjugation.

Example 7 Preparation of Pn-33F RAC/DMSO Conjugates to CRM₁₉₇

Preparation of Pn-33F RAC/DMSO Glycoconjugates

Compared to RAC/aqueous process, conjugation conducted via reductiveamination in an DMSO (RAC/DMSO) generally has a significantly lowerchance of de-O-acetylation. In view of the challenges associated withthe preservation of O-acetyl functionality using RAC/aqueous processdescribed in Example 6, an alternative approach using RAC/DMSO solvent,which has been successfully applied to produce pneumococcal conjugatevaccine (see e.g. WO 2006/110381) was evaluated.

Activated polysaccharide was compounded with sucrose (50% w/v in WFI)using a ratio of 25 grams of sucrose per gram of activatedpolysaccharide. The components were well mixed prior to shell freezingin dry ice/ethanol bath. The shell-frozen bottle of compounded mixturewas then lyophilized to dryness.

Lyophilized activated polysaccharide was reconstituted in dimethylsulfoxide (DMSO). DMSO was added to lyophilized CRM₁₉₇ forreconstitution. Reconstituted activated polysaccharide was combined withreconstituted CRM₁₉₇ in the reaction vessel. Conjugation was initiatedby adding NaCNBH3 to the reaction mixture. The reaction was incubated at23° C. for 20 hrs. Termination of the conjugation (capping) reaction wasachieved by adding NaBH4 and the reaction was continued for another 3hrs. The reaction mixture was diluted with 4-fold volume of 5 mMsuccinate-0.9% saline, pH 6.0 buffer and then filtered through 5 μmfilter prior to purification. Concentration and diafiltration of theconjugate was carried out using 100K MWCO membranes. Diafiltration wasperformed against 40-fold diavolume of 5 mM succinate-0.9% saline, pH6.0 buffer. The retentate was filtered through 0.45 and 0.22 μm filtersand analyzed.

Several conjugates were obtained using the above described process byvarying different parameters (e.g. saccharide-protein input ratio,reaction concentration, Meq of sodium cyanoborohydride, and watercontent). The overall data generated from conjugates prepared byRAC/DMSO process were demonstrated to be superior compared toRAC/aqueous process and allowed to prepare conjugates with goodconjugation yield, low % free saccharide (<5%) and higher degree ofconjugation (conjugated lysines). Additionally, it was possible topreserve more than 80% of acetyl functionality throughout the RAC/DMSOconjugation process.

Evaluation of Pn-33F RAC/DMSO Glycoconjugates Stability

Aliquots of representative RAC/DMSO conjugates prepared by the aboveprocess were dispensed into polypropylene tubes, which were storedeither at 4° C. or at 25° C. and stability was monitored for 3 monthsfor free saccharide. As shown at Table 10, the samples stored at 4° C.showed free saccharide increase by 4.8% in 3 months. However the samplesstored at 25° C. showed 15.4% increase in the % free saccharide in threemonths. The increase in % Free Saccharide in the RAC conjugates isattributed to the degradation of the conjugate, particularly at 25° C.

TABLE 10 Stability Results for RAC/DMSO Conjugate at 4° C. and 25° C.Time 0 3 wks 2 M 3 M Free Saccharide (%) at 4° C. 4.5 7.9 NA 9.3 FreeSaccharide (%) at 25° C. 4.5 12 15.7 19.9 wk = week; M = month.

The stability of another lot of RAC/DMSO conjugate was also studied at4° C., 25° C. and 37° C. Aliquots were dispensed into polypropylenetubes and monitored for potential trends in % free saccharide. As shownat Table 11 the samples stored at 4° C. showed 4.7% increase in % freesaccharide in 2 months. The increase in free saccharide wassignificantly higher at 25° C. and 37° C., indicating potentialdegradation of the conjugate.

TABLE 11 Stability Results for RAC/DMSO Conjugate at 4° C., 25° C. and37° C. Time 0 1 wk 2 wks 1 M 2 M Free Saccharide (%) at 4° C. 7.1 9.5 NANA 11.7 Free Saccharide (%) at 25° C. 7.1 9.3 12.7 14.5 NA FreeSaccharide (%) at 37° C. 7.1 14 19.1 23.6 NA wk = week; M = month.

Even though the conjugates generated by the RAC/DMSO process preservedthe O-Acetyl group, the increase in % free saccharide observed,particularly at 25° C. and above indicated potential instability usingthis route. In view of this observation of potential instability ofRAC/DMSO conjugates, RAC/DMSO was not seen as optimal for serotype 33Fconjugation and an alternative chemistry route was developed to generatemore stable conjugates (the eTEC conjugates).

Example 8 Preparation of Additional Pn-33F eTEC Conjugates

Additional Pn-33F eTEC Conjugates were generated using the processdescribed in Example 2. The reaction parameters and characterizationdata for these additional batches of Pn-33F eTEC glycoconjugates areshown in Table 12.

TABLE 12 Experimental Parameters and Characterization Data of furtherPn33F eTEC Conjugates Conjugate Batch 33F-8H 33F-9I 33F-10J 33F-11K33F-12L 33F-13M 33F-14N 33F-15O 33F-16P Activation level (mol of 0.22 0.11  0.11  0.13  0.14 0.13 0.06  0.13  0.11 thiol/mol ofpolysaccharide) Saccharide/Protein 0.75 0.8 0.8 0.8 0.8 0.8 0.8  0.8 0.8(Input) ratio Saccharide yield (%) 78%  88%  89%  67%  69% 86% 81% 91%88% Saccharide/Protein Ratio 1.0 2.2 2.1 1.4 1.4 1.4 2.2  1.9 1.9 FreeSaccharide <1% 6.8% 5.9% 2.3% 3.6% LOQ 8.2%  3.6%  6.6%  MW by SEC-MALLS(kDa) 4729 3293    3295    2246    2498    5539 3070    6009    3789   CMCA/CMC 6.6/LOQ 14.2/2.1 15.4/2.1 5.5/1 5.4/1.1 NA/LOQ 1.7/1.2 4.1/12.22.2/1.2 % Kd (≦0.3) 69% NA NA NA NA 88% 87% 87% 85% Acetylation level(mol of 0.86  0.93  0.87  1.01  0.99 0.71 0.78 0.8  0.82 acetate/mol ofpolysaccharide) LOQ = limit of quantitation.

As shown above and in Table 12, several Pn33F conjugates were obtainedusing the eTEC conjugation above. The eTEC chemistry allowed preparationof conjugates with high yield, low % free saccharide and high degree ofconjugation (conjugated lysines). Additionally, it was possible topreserve more than 80% of acetyl functionality using the eTECconjugation process.

Example 9 Evaluation of Pn-33F eTEC Glycoconjugates Stability: % FreeSaccharide Trends

Aliquots of conjugate batch 33F-2B (see table 2) were dispensed intopolypropylene tubes and stored at 4° C., 25° C., and 37° C.,respectively and monitored for trends in % free saccharide. The data (%free saccharide) are shown in Table 13. As shown in this Table, therewere no significant changes in the % free saccharide.

TABLE 13 % Free Saccharide Stability for Pn-33F eTEC Glycoconjugate at4° C., 25° C. and 37° C. Free Saccharide (%) Time Lot# 0 1 wk 3 wks 1 M2 M 3 M 6 M 4° C. 33F-2B 7.7 NA 8.3 NA 9.7 11.2 13 25° C. 7.7 NA 10.8 NA11.8 NA NA 37° C. 7.7 12.1 NA 13.4 NA NA NA wk = week; M = month.

The accelerated stability of another conjugate lot (Batch 33F-3C) wasalso conducted at 37° C. up to 1 month. As shown in Table 14, there wasno significant change to % free saccharide at 37° C., up to 1 month.

TABLE 14 % Free Saccharide Stability for Pn-33F eTEC Glycoconjugate at37° C. Free Saccharide (%) Time 0 1 day 1 wk 2 wks 1 M Lot# 37° C.33F-3C 4.4 5.9 6.4 7.1 7.2

To further confirm the stability of eTEC conjugates, additionalconjugate batches (33F-3C and 33F-5E (see Table 2 and Table 12)) storedat 4° C. were monitored up to approximately one year, for potentialtrends in % free saccharide. As shown in Table 15, there were nosignificant changes in % free saccharide levels for the conjugatesstored at 4° C. for an extended period up to approximately one year.

TABLE 15 % Free Saccharide Stability Results for Pn-33F eTECGlycoconjugates at 4° C. Free Saccharide (%) Time 0 3 M 4 M 12 M 14 MLot# 4° C. 33F-3C 4.4 NA 5.3 NA 7.6 33F-5E 7.3 6.3 NA 7.4 NA M = month

In contrast to the RAC/aqueous and RAC/DMSO conjugates, the Serotype 33Fconjugates generated by 33F eTEC chemistry were demonstrated to besignificantly more stable without noticeable degradation as monitored bythe free saccharide trends at various temperatures (real time andaccelerated).

Example 10 General Process for Preparation of(((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC) linked Glycoconjugate

Activation of Saccharide and Thiolation with Mercaptopropionylhydrazide(MPH) Spacer

The saccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO).Moisture content of the solution is determined by Karl Fischer (KF)analysis and adjusted to reach a moisture content of 0.1 and 0.4%,typically 0.2%.

To initiate the activation, a solution of1,1′-carbonyl-di-1,2,4-triazole (CDT) or 1,1′-carbonyldiimidazole (CDI)is freshly prepared at a concentration of 100 mg/mL in DMSO. Thesaccharide is activated with various amounts of CDT/CDI (1-10 molarequivalents) and the reaction is allowed to proceed for 1 hour at 23±2°C. The activation level may be determined by HPLC. MPH is freshlyprepared in anhydrous DMSO at a concentration of 50 mg/mL. The activatedsaccharide is reacted with 1-3 mol. eq. of MPH. The thiolation reactionis allowed to proceed for 21±2 hours at 23±2° C., to produce a thiolatedsaccharide. The thiolation level is determined by the added amount ofCDT/CDI.

Residual CDT/CDI in the activation reaction solution is quenched by theaddition of 100 mM sodium tetraborate, pH 9.0 solution. Calculations areperformed to determine the added amount of tetraborate and to adjust thefinal moisture content to be up to 1-2% of total aqueous.

Purification of Activated Thiolated Saccharide

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 1-5 mol. eq., is added and allowedto proceed for 3±1 hours at 23±2° C. The reaction mixture is thendiluted 5-fold by addition to pre-chilled 5 mM sodium succinate in 0.9%saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltration ofthiolated saccharide is performed using 40-fold diavolume of pre-chilled10 mM sodium phosphate monobasic, pH 4.3. An aliquot of activatedthiolated saccharide retentate is pulled to determine the saccharideconcentration and thiol content (Ellman) assays.

Activation and Purification of Bromoacetylated Carrier Protein

Free amino groups of the carrier protein are bromoacteylated by reactionwith a bromoacetylating agent, such as bromoacetic acidN-hydroxysuccinimide ester (BAANS), bromoacetylbromide, or anothersuitable reagent.

The carrier protein (in 0.1M Sodium Phosphate, pH 8.0±0.2) is first keptat 8±3° C., at about pH 7 prior to activation. To the protein solution,the N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a stockdimethylsulfoxide (DMSO) solution (20 mg/mL) is added in a ratio of0.25-0.5 BAANS: protein (w/w). The reaction is gently mixed at 5±3° C.for 30-60 minutes. The resulting bromoacetylated (activated) protein ispurified, e.g., by ultrafiltration/diafiltration using 10 kDa MWCOmembrane using 10 mM phosphate (pH 7.0) buffer. Following purification,the protein concentration of the bromoacetylated carrier protein isestimated by Lowry protein assay.

The extent of activation is determined by total bromide assay byion-exchange liquid chromatography coupled with suppressed conductivitydetection (ion chromatography). The bound bromide on the activatedbromoacetylated protein is cleaved from the protein in the assay samplepreparation and quantitated along with any free bromide that may bepresent. Any remaining covalently bound bromine on the protein isreleased by conversion to ionic bromide by heating the sample inalkaline 2-mercaptoethanol

Activation and Purification of Bromoacetylated CRM₁₉₇

CRM₁₉₇ is diluted to 5 mg/mL with 10 mM phosphate buffered 0.9% NaCl pH7 (PBS) and then made 0.1 M NaHCO₃ pH 7.0 using 1 M stock solution.BAANS is added at a CRM₁₉₇: BAANS ratio 1:0.35 (w:w) using a BAANS stocksolution of 20 mg/mL DMSO. The reaction mixture is incubated at between3° C. and 11° C. for 30 mins-1 hour then purified byultrafiltration/diafiltration using a 10K MWCO membrane and 10 mM SodiumPhosphate/0.9% NaCl, pH 7.0. The purified activated CRM₁₉₇ is assayed bythe Lowry assay to determine the protein concentration and then dilutedwith PBS to 5 mg/mL. Sucrose is added to 5% wt/vol as a cryoprotectantand the activated protein is frozen and stored at −25° C. until neededfor conjugation.

Bromoacetylation of lysine residues of CRM₁₉₇ is very consistent,resulting in the activation of 15 to 25 lysines from 39 lysinesavailable. The reaction produced high yields of activated protein.

Conjugation of Activated Thiolated Saccharide to Bromoacetylated CarrierProtein

Before starting the conjugation reaction, the reaction vessels arepre-cooled to 5±3° C. Bromoacetylated carrier protein and activatedthiolated saccharide are subsequently added and mixed at an agitationspeed of 150-200 rpm. The saccharide/protein input ratio is 0.9±0.1. Thereaction pH is adjusted to 8.0±0.1 with 1 M NaOH solution. Theconjugation reaction is allowed to proceed at 5±3° C. for 20±2 hours.

Capping of Residual Reactive Functional Groups

The unreacted bromoacetylated residues on the carrier protein arequenched by reacting with 2 mol. eq. of N-Acetyl-L-Cysteinehydrochloride as a capping reagent for 3 hours at 5±3° C. Residual freesulfhydryl groups are capped with 4 mol. eq. of iodoacetamide (IAA) for20 hours at 5±3° C.

Purification of Oxo-eTAC-Linked Glycoconjugate

The conjugation reaction (post-IAA-capped) mixture is filtered through0.45 μm filter. Ultrafiltration/dialfiltration of the glycoconjugate isperformed against 5 mM succinate-0.9% saline, pH 6.0. The glycoconjugateretentate is then filtered through 0.2 μm filter. An aliquot ofglycoconjugate is pulled for assays. The remaining glycoconjugate isstored at 5±3° C.

Example 11 Preparation of (((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC)linked Glycoconjugate through Activation of Saccharide and Thiolationwith L-Cystine Dimethylester Dihydrochloride Spacer

The saccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO).Moisture content of the solution is determined by Karl Fischer (KF)analysis and adjusted to reach a moisture content of 0.1 and 0.4%,typically 0.2%.

To initiate the activation, a solution of1,1′-carbonyl-di-1,2,4-triazole (CDT) or 1,1′-carbonyldiimidazole (CDI)is freshly prepared at a concentration of 100 mg/mL in DMSO. Thesaccharide is activated with various amounts of CDT/CDI (1-10 molarequivalents) and the reaction is allowed to proceed for 1 hour at 23±2°C. The activation level may be determined by HPLC. L-Cystinedimethylester dihydrochloride is freshly prepared in anhydrous DMSO at aconcentration of 50 mg/mL. The activated saccharide is reacted with 1-2mol. eq. of L-Cystine dimethylester dihydrochloride. The thiolationreaction is allowed to proceed for 21±2 hours at 23±2° C., to produce athiolated saccharide. The thiolation level is determined by the addedamount of CDT/CDI.

Residual CDT/CDI in the activation reaction solution is quenched by theaddition of 100 mM sodium tetraborate, pH 9.0 solution. Calculations areperformed to determine the added amount of tetraborate and to adjust thefinal moisture content to be up to 1-2% of total aqueous.

Purification of Activated Thiolated Saccharide

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 5-10 mol. eq., is added andallowed to proceed for 3±1 hours at 23±2° C. The reaction mixture isthen diluted 5-fold by addition to pre-chilled 5 mM sodium succinate in0.9% saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltrationof thiolated saccharide is performed using 40-fold diavolume ofpre-chilled 10 mM sodium phosphate monobasic, pH 4.3. An aliquot ofactivated thiolated saccharide retentate is pulled to determine thesaccharide concentration and thiol content (Ellman) assays.

Conjugation of Activated Thiolated Saccharide to Bromoacetylated CarrierProtein, Capping and purification of the eTAC conjugate follows thegeneral procedure described in example 10, as above.

Example 12 Preparation of (((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC)linked Glycoconjugate through activation of Saccharide and Thiolationwith 2-(2-Aminoethoxy)Ethane-1-Thiol (AEET) Spacer

The saccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO).Moisture content of the solution is determined by Karl Fischer (KF)analysis and adjusted to reach a moisture content of 0.1 and 0.4%,typically 0.2%.

To initiate the activation, a solution of1,1′-carbonyl-di-1,2,4-triazole (CDT) or 1,1′-carbonyldiimidazole (CDI)is freshly prepared at a concentration of 100 mg/mL in DMSO. Thesaccharide is activated with various amounts of CDT/CDI (1-10 molarequivalents) and the reaction is allowed to proceed for 1 hour at 23±2°C. The activation level may be determined by HPLC. AEET is freshlyprepared in anhydrous DMSO at a concentration of 50 mg/mL. The activatedsaccharide is reacted with 1-2 mol. eq. of AEET. The thiolation reactionis allowed to proceed for 21±2 hours at 23±2° C., to produce a thiolatedsaccharide. The thiolation level is determined by the added amount ofCDT/CDI.

Residual CDT/CDI in the activation reaction solution is quenched by theaddition of 100 mM sodium tetraborate, pH 9.0 solution. Calculations areperformed to determine the added amount of tetraborate and to adjust thefinal moisture content to be up to 1-2% of total aqueous.

Purification of Activated Thiolated Saccharide

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 1-5 mol. eq., is added and allowedto proceed for 3±1 hours at 23±2° C. The reaction mixture is thendiluted 5-fold by addition to pre-chilled 5 mM sodium succinate in 0.9%saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltration ofthiolated saccharide is performed using 40-fold diavolume of pre-chilled10 mM sodium phosphate monobasic, pH 4.3. An aliquot of activatedthiolated saccharide retentate is pulled to determine the saccharideconcentration and thiol content (Ellman) assays.

Conjugation of Activated Thiolated Saccharide to Bromoacetylated CarrierProtein, Capping and purification of the eTAC conjugate follows thegeneral procedure described in example 10, as above.

Example 13 Preparation of (((2-Oxoethyl)Thio)Alkyl)Carbamate (Oxo-eTAC)Linked Glycoconjugate Through Activation of Saccharide and Thiolationwith 4-Amino-1-Butanethiol Hydrochloride Spacer

The saccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO).Moisture content of the solution is determined by Karl Fischer (KF)analysis and adjusted to reach a moisture content of 0.1 and 0.4%,typically 0.2%.

To initiate the activation, a solution of1,1′-carbonyl-di-1,2,4-triazole (CDT) or 1,1′-carbonyldiimidazole (CDI)is freshly prepared at a concentration of 100 mg/mL in DMSO. Thesaccharide is activated with various amounts of CDT/CDI (1-10 molarequivalents) and the reaction is allowed to proceed for 1 hour at 23±2°C. The activation level may be determined by HPLC. 4-Amino-1-butanethiolhydrochloride is freshly prepared in anhydrous DMSO at a concentrationof 50 mg/mL. The activated saccharide is reacted with 1-2 mol. eq. of4-Amino-1-butanethiol hydrochloride. The thiolation reaction is allowedto proceed for 21±2 hours at 23±2° C., to produce a thiolatedsaccharide. The thiolation level is determined by the added amount ofCDT/CDI.

Residual CDT/CDI in the activation reaction solution is quenched by theaddition of 100 mM sodium tetraborate, pH 9.0 solution. Calculations areperformed to determine the added amount of tetraborate and to adjust thefinal moisture content to be up to 1-2% of total aqueous.

Purification of Activated Thiolated Saccharide

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 1-5 mol. eq., is added and allowedto proceed for 3±1 hours at 23±2° C. The reaction mixture is thendiluted 5-fold by addition to pre-chilled 5 mM sodium succinate in 0.9%saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltration ofthiolated saccharide is performed using 40-fold diavolume of pre-chilled10 mM sodium phosphate monobasic, pH 4.3. An aliquot of activatedthiolated saccharide retentate is pulled to determine the saccharideconcentration and thiol content (Ellman) assays.

Conjugation of Activated Thiolated Saccharide to Bromoacetylated CarrierProtein, Capping and purification of the eTAC conjugate follows thegeneral procedure described in example 10, as above.

Example 14 Preparation of Pn-33F Oxo-eTAC Conjugates UsingMercaptopropionylhydrazide (MPH) Activation Process

Activation of Pn33F Polysaccharide with Mercaptopropionylhydrazide (MPH)Spacer

Pn-33F polysaccharide is compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture is shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized 33F polysaccharide is reconstituted inanhydrous dimethylsulfoxide (DMSO). Moisture content of the lyophilized33F/DMSO solution is determined by Karl Fischer (KF) analysis. Themoisture content is adjusted by adding WFI to the 33F/DMSO solution toreach a moisture content of 0.2%.

To initiate the activation, 1,1′-carbonyl-di-1,2,4-triazole (CDT) isfreshly prepared as 100 mg/mL in DMSO solution. Pn33F polysaccharide isactivated with various amounts of CDT prior to the thiolation step. TheCDT activation is carried out at 23±2° C. for 1 hour. The activationlevel is determined by HPLC (A220/A205). Sodium tetraborate, 100 mM, pH9.0 solution is added to quench any residual CDT in the activationreaction solution. Calculations are performed to determine the addedamount of tetraborate and to allow the final moisture content to be 1.2%of total aqueous. The reaction is allowed to proceed for 1 hour at 23±2°C.

Thiolation of Activated Pn-33F Polysaccharide

MPH is freshly prepared in anhydrous DMSO and 1-2 mol. eq. of MPH isadded to the activated polysaccharide reaction solution. The reaction isallowed to proceed for 21±2 hours at 23±2° C. To the reaction mixture asolution of tris(2-carboxyethyl)phosphine (TCEP), 1-5 mol. eq., is addedand allowed to proceed for 3±1 hours at 23±2° C. The reaction mixture isthen diluted 5-fold by addition to pre-chilled 5 mM sodium succinate in0.9% saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltrationof thiolated saccharide is performed using 40-fold diavolume ofpre-chilled 10 mM sodium phosphate monobasic, pH 4.3 with 100K MWCOultrafilter membrane cassettes. The thiolated 33F polysaccharideretentate is pulled for both saccharide concentration and thiol (Ellman)assays.

Conjugation Process

Conjugation of Thiolated Pn33F Polysaccharide to Bromoacetylated CRM₁₉₇

The CRM₁₉₇ carrier protein is activated separately by bromoacetylation,and then reacted with the activated Pn-33F polysaccharide for theconjugation reaction. Before starting the conjugation reaction, thereaction vessel is pre-cooled to 5±3° C. Bromoacetylated CRM₁₉₇ andthiolated 33F polysaccharide were mixed together in a reaction vessel atan agitation speed of 150-200 rpm. The saccharide/protein input ratio is0.9±0.1. The reaction pH is adjusted to 8.0-9.0. The conjugationreaction is allowed to proceed at 5±3° C. for 20±2 hours.

Capping of Reactive Groups on Bromoacetylated CRM₁₉₇ and Thiolated Pn33FPolysaccharide

The unreacted bromoacetylated residues on CRM₁₉₇ proteins were capped byreacting with 2 mol. eq. of N-Acetyl-L-Cysteine hydrochloride for 3hours at 5±3° C., followed by capping any residual free sulfhydrylgroups of the thiolated 33F-polysaccharide with 4 mol. eq. ofiodoacetamide (IAA) for 20 hours at 5±3° C.

Purification of Oxo-eTAC-Linked Pn-33F Glycoconjugate

The conjugation solution is filtered through a 0.45 μm or 5 μm filter.Dialfiltration of the 33F glycoconjugate is carried out with 300K MWCOultrafilter membrane cassettes. Diafiltration is performed against 5 mMsuccinate-0.9% saline, pH 6.0. The Pn-33F glycoconjugate 300K retentateis then filtered through a 0.22 μm filter and stored at 5±3° C.

Example 15 Preparation of Pn-33F Oxo-eTAC Conjugates Using L-CystineDimethylester Activation Process

Activation of Pn33F Polysaccharide with L-Cystine DimethylesterDihydrochloride Spacer

Pn-33F polysaccharide was compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture was shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized 33F polysaccharide was reconstituted inanhydrous dimethylsulfoxide (DMSO). Moisture content of the lyophilized33F/DMSO solution was determined by Karl Fischer (KF) analysis. Themoisture content was adjusted by adding WFI to the 33F/DMSO solution toreach a moisture content of 0.2%.

To initiate the activation, 1,1′-carbonyl-di-1,2,4-triazole (CDT) wasfreshly prepared as 100 mg/mL in DMSO solution. Pn33F polysaccharide wasactivated with various amounts of CDT prior to the thiolation step. TheCDT activation was carried out at 23±2° C. for 1 hour. The activationlevel was determined by HPLC (A220/A205). Sodium tetraborate, 100 mM, pH9.0 solution was added to quench any residual CDT in the activationreaction solution. Calculations were performed to determine the addedamount of tetraborate and to allow the final moisture content to be 1.2%of total aqueous. The reaction was allowed to proceed for 1 hour at23±2° C.

Thiolation of Activated Pn-33F Polysaccharide

L-Cystine dimethylester dihydrochloride was freshly prepared inanhydrous DMSO and 1 mol. eq. of L-Cystine dimethylester dihydrochloridewas added to the activated polysaccharide reaction solution. Thereaction was allowed to proceed for 21±2 hours at 23±2° C. The thiolatedsaccharide solution was diluted 10-fold by addition to pre-chilled 5 mMsodium succinate in 0.9% saline, pH 6.0. The diluted reaction solutionwas filtered through a 5 μm filter. Dialfiltration of thiolated Pn-33Fpolysaccharide was carried out with 100K MWCO ultrafilter membranecassettes, using Water for Injection (WFI).

Reduction and Purification of Activated Thiolated Pn-33F Polysaccharide

To the retentate a solution of tris(2-carboxyethyl)phosphine (TCEP), 5mol. eq., was added after dilution by 10% volume of 0.1M sodiumphosphate buffer, pH 6.0. This reduction reaction was allowed to proceedfor 2±1 hours at 23±2° C. Dialfiltration of thiolated 33F polysaccharidewas carried out with 100K MWCO ultrafilter membrane cassettes.Diafiltration was performed against pre-chilled 10 mM sodium phosphate,pH 4.3. The thiolated 33F polysaccharide retentate was pulled for bothsaccharide concentration and thiol (Ellman) assays.

Alternative Reduction and Purification of Activated Thiolated Pn-33FPolysaccharide

As an alternative to the purification procedure described above, 33Factivated thiolated saccharide was also purified as follows.

To the thiolated saccharide reaction mixture a solution oftris(2-carboxyethyl)phosphine (TCEP), 5 mol. eq., was added and allowedto proceed for 3±1 hours at 23±2° C. The reaction mixture was thendiluted 5-fold by addition to pre-chilled 5 mM sodium succinate in 0.9%saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltration ofthiolated saccharide was performed using 40-fold diavolume ofpre-chilled 10 mM sodium phosphate monobasic, pH 4.3 with 100K MWCOultrafilter membrane cassettes. The thiolated 33F polysaccharideretentate was pulled for both saccharide concentration and thiol(Ellman) assays.

Conjugation Process

Conjugation of Thiolated Pn33F Polysaccharide to Bromoacetylated CRM₁₉₇

The CRM₁₉₇ carrier protein was activated separately by bromoacetylation,and then reacted with the activated Pn-33F polysaccharide for theconjugation reaction. Before starting the conjugation reaction, thereaction vessel was pre-cooled to 5±3° C. Bromoacetylated CRM₁₉₇ andthiolated 33F polysaccharide were mixed together in a reaction vessel atan agitation speed of 150-200 rpm. The saccharide/protein input ratiowas 0.9±0.1. The reaction pH was adjusted to 8.0-9.0. The conjugationreaction was allowed to proceed at 5±3° C. for 20±2 hours.

Capping of Reactive Groups on Bromoacetylated CRM₁₉₇ and Thiolated Pn33FPolysaccharide

The unreacted bromoacetylated residues on CRM₁₉₇ proteins were capped byreacting with 2 mol. eq. of Cysteamine hydrochloride for 3 hours at 5±3°C., followed by capping any residual free sulfhydryl groups of thethiolated 33F-polysaccharide with 4 mol. eq. of iodoacetamide (IAA) for20 hours at 5±3° C.

Purification of Oxo-eTAC-Linked Pn-33F Glycoconjugate

The conjugation solution was filtered through a 0.45 μm or 5 μm filter.Dialfiltration of the 33F glycoconjugate was carried out with 300K MWCOultrafilter membrane cassettes. Diafiltration was performed against 5 mMsuccinate-0.9% saline, pH 6.0. The Pn-33F glycoconjugate 300K retentatewas then filtered through a 0.22 μm filter and stored at 5±3° C.

Results

Characterization and process data for representative Pn-33F oxo-eTACconjugates using L-Cystine dimethylester dihydrochloride spacer areprovided at Table 16

Example 16 Preparation of Pn-33F oxo-eTAC Conjugates using2-(2-Aminoethoxy)Ethane-1-Thiol (AEET) Activation Process

Activation of Pn33F Polysaccharide with 2-(2-Aminoethoxy)Ethane-1-Thiol(AEET) Spacer

Pn-33F polysaccharide was compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture was shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized 33F polysaccharide was reconstituted inanhydrous dimethylsulfoxide (DMSO). Moisture content of the lyophilized33F/DMSO solution was determined by Karl Fischer (KF) analysis. Themoisture content was adjusted by adding WFI to the 33F/DMSO solution toreach a moisture content of 0.2%.

To initiate the activation, 1,1′-carbonyl-di-1,2,4-triazole (CDT) wasfreshly prepared as 100 mg/mL in DMSO solution. Pn33F polysaccharide wasactivated with various amounts of CDT prior to the thiolation step. TheCDT activation was carried out at 23±2° C. for 1 hour. The activationlevel is determined by HPLC (A220/A205). Sodium tetraborate, 100 mM, pH9.0 solution was added to quench any residual CDT in the activationreaction solution. Calculations were performed to determine the addedamount of tetraborate and to allow the final moisture content to be 1.2%of total aqueous. The reaction was allowed to proceed for 1 hour at23±2° C.

Thiolation of Activated Pn-33F Polysaccharide

AEET was freshly prepared in anhydrous DMSO and 1-2 mol. eq. of AEET wasadded to the activated polysaccharide reaction solution. The reactionwas allowed to proceed for 21±2 hours at 23±2° C. To the reactionmixture a solution of tris(2-carboxyethyl)phosphine (TCEP), 1-5 mol.eq., was added and allowed to proceed for 3±1 hours at 23±2° C. Thereaction mixture was then diluted 5-fold by addition to pre-chilled 5 mMsodium succinate in 0.9% saline, pH 6.0 and filtered through a 5 μmfilter. Dialfiltration of thiolated saccharide was performed using40-fold diavolume of pre-chilled 10 mM sodium phosphate monobasic, pH4.3 with 100K MWCO ultrafilter membrane cassettes. The thiolated 33Fpolysaccharide retentate was pulled for both saccharide concentrationand thiol (Ellman) assays.

Conjugation Process

Conjugation of Thiolated Pn33F Polysaccharide to Bromoacetylated CRM₁₉₇

The CRM₁₉₇ carrier protein was activated separately by bromoacetylation,and then reacted with the activated Pn-33F polysaccharide for theconjugation reaction. Before starting the conjugation reaction, thereaction vessel was pre-cooled to 5±3° C. Bromoacetylated CRM₁₉₇ andthiolated 33F polysaccharide were mixed together in a reaction vessel atan agitation speed of 150-200 rpm. The saccharide/protein input ratiowas 0.9±0.1. The reaction pH is adjusted to 8.0-9.0. The conjugationreaction was allowed to proceed at 5±3° C. for 20±2 hours.

Capping of Reactive Groups on Bromoacetylated CRM₁₉₇ and Thiolated Pn33FPolysaccharide

The unreacted bromoacetylated residues on CRM₁₉₇ proteins were capped byreacting with 2 mol. eq. of N-Acetyl-L-Cysteine hydrochloride for 3hours at 5±3° C., followed by capping any residual free sulfhydrylgroups of the thiolated 33F-polysaccharide with 4 mol. eq. ofiodoacetamide (IAA) for 20 hours at 5±3° C.

Purification of Oxo-eTAC-Linked Pn-33F Glycoconjugate

The conjugation solution was filtered through a 0.45 μm or 5 μm filter.Dialfiltration of the 33F glycoconjugate was carried out with 300K MWCOultrafilter membrane cassettes. Diafiltration was performed against 5 mMsuccinate-0.9% saline, pH 6.0. The Pn-33F glycoconjugate 300K retentatewas then filtered through a 0.22 μm filter and stored at 5±3° C.

Results

Characterization and process data for representative Pn-33F oxo-eTACconjugates using 2-(2-aminoethoxy)ethane-1-thiol (AEET) spacer areprovided at Table 16.

Example 17 Preparation of Pn-33F Oxo-eTAC Conjugates Using4-Amino-1-Butanethiol (ABT) Activation Process

Activation of Pn33F Polysaccharide with 4-Amino-1-ButanethiolHydrochloride Spacer

Pn-33F polysaccharide was compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture was shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized 33F polysaccharide was reconstituted inanhydrous dimethylsulfoxide (DMSO). Moisture content of the lyophilized33F/DMSO solution was determined by Karl Fischer (KF) analysis. Themoisture content was adjusted by adding WFI to the 33F/DMSO solution toreach a moisture content of 0.2%.

To initiate the activation, 1,1′-carbonyl-di-1,2,4-triazole (CDT) wasfreshly prepared as 100 mg/mL in DMSO solution. Pn33F polysaccharide wasactivated with various amounts of CDT prior to the thiolation step. TheCDT activation was carried out at 23±2° C. for 1 hour. The activationlevel was determined by HPLC (A220/A205). Sodium tetraborate, 100 mM, pH9.0 solution was added to quench any residual CDT in the activationreaction solution. Calculations were performed to determine the addedamount of tetraborate and to allow the final moisture content to be 1.2%of total aqueous. The reaction was allowed to proceed for 1 hour at23±2° C.

Thiolation of Activated Pn-33F Polysaccharide

4-Amino-1-butanethiol hydrochloride was freshly prepared in anhydrousDMSO and 1-2 mol. eq. of 4-Amino-1-butanethiol hydrochloride was addedto the activated polysaccharide reaction solution. The reaction wasallowed to proceed for 21±2 hours at 23±2° C. To the reaction mixture asolution of tris(2-carboxyethyl)phosphine (TCEP), 1-5 mol. eq., wasadded and allowed to proceed for 3±1 hours at 23±2° C. The reactionmixture was then diluted 5-fold by addition to pre-chilled 5 mM sodiumsuccinate in 0.9% saline, pH 6.0 and filtered through a 5 μm filter.Dialfiltration of thiolated saccharide was performed using 40-folddiavolume of pre-chilled 10 mM sodium phosphate monobasic, pH 4.3 with100K MWCO ultrafilter membrane cassettes. The thiolated 33Fpolysaccharide retentate was pulled for both saccharide concentrationand thiol (Ellman) assays.

Conjugation Process

Conjugation of Thiolated Pn33F Polysaccharide to Bromoacetylated CRM₁₉₇

The CRM₁₉₇ carrier protein was activated separately by bromoacetylation,and then reacted with the activated Pn-33F polysaccharide for theconjugation reaction. Before starting the conjugation reaction, thereaction vessel was pre-cooled to 5±3° C. Bromoacetylated CRM₁₉₇ andthiolated 33F polysaccharide were mixed together in a reaction vessel atan agitation speed of 150-200 rpm. The saccharide/protein input ratio is0.9±0.1. The reaction pH was adjusted to 8.0-9.0. The conjugationreaction was allowed to proceed at 5±3° C. for 20±2 hours.

Capping of Reactive Groups on Bromoacetylated CRM₁₉₇ and Thiolated Pn33FPolysaccharide

The unreacted bromoacetylated residues on CRM₁₉₇ proteins were capped byreacting with 2 mol. eq. of N-Acetyl-L-Cysteine hydrochloride for 3hours at 5±3° C., followed by capping any residual free sulfhydrylgroups of the thiolated 33F-polysaccharide with 4 mol. eq. ofiodoacetamide (IAA) for 20 hours at 5±3° C.

Purification of Oxo-eTAC-Linked Pn-33F Glycoconjugate

The conjugation solution was filtered through a 0.45 μm or 5 μm filter.Dialfiltration of the 33F glycoconjugate was carried out with 300K MWCOultrafilter membrane cassettes. Diafiltration was performed against 5 mMsuccinate-0.9% saline, pH 6.0. The Pn-33F glycoconjugate 300K retentatewas then filtered through a 0.22 μm filter and stored at 5±3° C.

Results

Characterization and process data for representative Pn-33F oxo-eTACconjugates using 4-Amino-1-butanethiol hydrochloride (ABT) spacer areprovided at Table 16.

TABLE 16 Experimental Parameters and Characterization Data for Pn-33FeTAC conjugates with various types of linkers Conjugate BatchPn-33F-eTAC- Pn-33F-eTAC- Pn-33F-eTAC- AEET (see Cystine (see ABT (seeexample 16) example 15) example 17) Polysaccharide Mw 1565 kDa 1565 kDa1565 kDa (SEC-MALLS) Poly Activation Meq. of CDT 3 3 3 Linker AEETCystine ABT MEq. of Linker 1 1 1 MEq. of TCEP   5 meq   5 meq   5 meqYield 80% 84% 75% Thiol content (mol 0.19 0.033 0.18 of thiol/mol ofpolysaccharide) Conjugation to CRM₁₉₇ Input ratio 0.80 0.80 0.80Conjugate Results Saccharide yield (%) 55% 52% 52% Saccharide/Protein1.6 2.1 2.6 Ratio Free Saccharide  8%  7%  7% Free Protein <1% <1% <1%Conjugate Mw 4518 kDa 4369 kDa 3616 kDa (SEC-MALS)

Polysaccharide-protein conjugates were generated with each of theselinkers having good quality attributes including molecular weight, highyield, low free saccharide and low free protein levels.

Example 18 General Process for Preparation of(((2-Oxoethyl)Thio)Alkyl)Amine (Oxo-eTAAN) Linked Glycoconjugate ViaPrimary Hydroxyl Oxidation Oxidation of Primary Hydroxyl Groups ofPolysaccharide

Oxidation of primary alcohols in polysaccharide was achieved using theTEMPO/NCS system in either aqueous or organic solvent. Thepolysaccharides were oxidized to varying degrees of oxidation (DO)levels by adjusting the amount of NCS cooxidant. Generally 0.5-2.5 MolarEquivalents of NCS was used to achieve the target Degree of Oxidation.The oxidation reaction was typically done in bicarbonate/carbonatebuffer (0.5 M NaHCO₃/0.05 M Na₂CO₃ buffer, pH 8.6) or sodium phosphatebuffers of pH 6.5, 7.0, 7.5 and 8.0 and is complete in 2 hours. In someactivation experiments a primary alcohol such as n-propanol was used toquench the reagents in order to avoid saccharide overoxidation.

In a specific example, the Serotype 33F polysaccharide was added to areaction vessel at a concentration of 4.0 mg/mL and mixed withbicarbonate/carbonate buffer (0.5 M NaHCO₃/0.05 M Na₂CO₃ buffer, pH 8.6)at a ratio of 1:1 v/v. To the stirred mixture was added ≦0.1 molequivalent of TEMPO. The reaction was started by the addition of 0.6 to1.0 mol equivalent of N-chlorosuccinimide (NCS). The reaction mixturewas stirred at room temperature for 2 hours, after which the activatedpolysaccharide was purified by diafiltration, with WFI using a 30Kultrafiltration membrane. The purified polysaccharide was collected andthe degree of oxidation (DO) was determined by quantitative measurementsof aldehyde (using a 3-methyl-2-benothiazolinone hydrazone (MBTH) assay)and polysaccharide (using an anthrone assay).

Thiolation of Oxidized Polysaccharide Using Reductive AminationChemistry Activation Using 2-(2-Aminoethoxy)Ethane-1-Thiol (AEET) Spacer(3^(rd) Column of Table 17)

The oxidized and purified Serotype 33F polysaccharide was added to areaction vessel followed by the addition of 0.5 M Sodium phosphatebuffer (pH 6.5) to a final buffer concentration of 0.1 M. To thissolution, 1 Meq 2-(2-aminoethoxy)ethane-1-thiol(NH₂—CH₂—CH₂—O—CH₂—CH₂—SH) (AEET) was added and mixed thoroughly inorder to obtain a homogenous solution. The pH was adjusted to 6.8 usingdiluted HCl or IN NaOH solution. This was followed by the addition of1.5 molar equivalents of NaCNBH₃. The reaction mixture was stirred for48 hours at room temperature (23° C.). The reaction mixture was thendiluted with 1×0.9% saline and the unreacted aldehyde groups were“capped” with 2 molar equivalents of sodium borohydride. The sodiumborohydride also functions as a reducing agent for the reduction of anydisulfide formed during the reaction. The capping reaction time wastypically 3 hours.

Activation Using Cystamine Spacer (2^(nd) Column of Table 17)

The oxidized and purified Serotype 33F polysaccharide was added to areaction vessel followed by the addition of 0.5 M Sodium phosphatebuffer (pH 6.5) to a final buffer concentration of 0.1 M. To thissolution, 1 MEq of Cystamine dihydrochloride solution was added andmixed thoroughly in order to obtain a homogenous solution. The pH wasadjusted to 6.8 using diluted HCl or IN NaOH solution. This was followedby the addition of 1.5 molar equivalents of NaCNBH₃. The reactionmixture was stirred for 48 hours at room temperature (23° C.).Alternatively the activated saccharide was reacted with 1 MEq ofcysteamine hydrochloride. The reaction mixture was then diluted with1×0.9% saline and the unreacted aldehyde groups were “capped” with 2molar equivalents of sodium borohydride. The sodium borohydride alsofunctions as a reducing agent for the reduction of any disulfide formedduring the reaction. The capping reaction time was typically 3 hours.

Purification of the Activated Thiolated Saccharide

Any remaining disulfide bonds present in the saccharide was reducedusing a suitable reducing agent such as (2-carboxyethyl)phosphine (TCEP)or dithioerythritol (DTT). The thiolated saccharide reaction mixture wasdiluted 10-fold by addition to pre-chilled 5 mM sodium succinate in 0.9%saline, pH 6.0 and filtered through a 5 μm filter. Dialfiltration ofthiolated saccharide was performed against 40-fold diavolume of WFI. Tothe retentate a solution of tris(2-carboxyethyl)phosphine (TCEP), 1-5mol. eq., was added after dilution by 10% volume of 0.1M sodiumphosphate buffer, pH 6.0. This reduction reaction was allowed to proceedfor 20±2 hours at 5±3° C. Purification of the activated thiolatedsaccharide was performed preferably by ultrafiltration/dialfiltration ofagainst pre-chilled 10 mM sodium phosphate monobasic, pH 4.3.Alternatively, the thiolated saccharide was purified by standard sizeexclusion chromatographic (SEC) procedures or ion exchangechromatographic methods. An aliquot of activated thiolated saccharideretentate was pulled to determine the saccharide concentration and thiolcontent (Ellman) assays.

In another example, low molecular weight saccharides are used to reactwith the aminothiol reagent utilizing the reducing end of the saccharidechain. The low molecular weight saccharides are prepared by eitherchemical hydrolysis or mechanical homogenization of high molecularweight polysaccharides.

Conjugation of Thiolated 33F with Bromoacetylated CRM₁₉₇

Before starting the conjugation reaction, the reaction vessels werepre-cooled to 5±3° C. Bromoacetylated carrier protein and activatedthiolated saccharide were subsequently added and mixed at an agitationspeed of 150-200 rpm. The saccharide/protein input ratio was 0.9±0.1.The reaction pH was adjusted to 8.0±0.1 with 1 M NaOH solution. Theconjugation reaction was allowed to proceed at 5° C. for 20±2 hours. Theunreacted bromoacetylated residues on the carrier protein were quenchedby reacting with 2 mol. eq. of N-acetyl-L-cysteine as a capping reagentfor 3 hours at 5±3° C. Residual free sulfhydryl groups were capped with4 mol. eq. of iodoacetamide (IAA) for 20 hours at 5±3° C.

The conjugation reaction (post-IAA-capped) mixture was filtered througha 5 μm filter and then purified using 100K MWCO ultra filtrationmembranes against 5 mM succinate-0.9% saline, pH 6.0. The purifiedconjugate was then filtered through 0.45/0.22 μm filters to obtain thebulk conjugate.

Results

Characterization and process data for representative Pn-33F eTAAN usingeither 2-(2-aminoethoxy)ethane-1-thiol (AEET) or cystamine as spacer andconjugated to CRM₁₉₇ are provided at Table 17.

TABLE 17 Experimental Parameters and Characterization Data for Pn-33FeTAAN conjugates Conjugate Batch Pn-33F-eTAAN- Pn-33F-eTAAN- TEMPO-TEMPO- Cystamine AEET Polysaccharide Mw 1565 kDa  1565 kDa  (SEC-MALLS)Poly Oxidation TEMPO (% v/v) 1 1 NCS (mol. eq.) 0.6 0.6 Yield 68% 68%Oxidized poly Mw (SEC-MALS) 217 kDa 217 kDa Degree of Oxidation 10 10Thiolation Linker Cystamine AEET NaCNBH3 (mol. eq.) 1.5 1.5 Yield 19%33% Thiol content (mol of thiol/mol of 0.09 0.09 polysaccharide)Conjugation to CRM197 Input ratio (SPR) 0.8 0.8 Conjugate ResultsSaccharide yield (%) 89% 80% Saccharide/Protein Ratio 0.96 0.85 FreeSaccharide 22% 13% Conjugate Mw (SEC-MALLS) 331 kDa 512 kDa

Polysaccharide-protein conjugates were generated with each of theselinkers having good quality attributes including molecular weight, highyield, low free saccharide levels.

Example 19 General Process for Preparation of(((2-Oxoethyl)Thio)Alkyl)Amine (Oxo-eTAAN) linked Glycoconjugate ViaOxidation of Vicinal Diols of Polysaccharide

Alternatively the saccharides can be activated by oxidizing the vicinalcis diols using periodate. In a specific example, the serotype 33Fpolysaccharide was dissolved in 100 mM sodium phosphate buffer (pH 6.0)to a final concentration of 2 mg/ml in 25 mM buffer. The reaction wasinitiated by the addition of 0.2 Meq of sodium periodate (50 mg/mLsolution in water). After 18 hours the activated polysaccharide waspurified by diafiltration, with WFI using a 30K ultrafiltrationmembrane. The purified polysaccharide was collected and the degree ofoxidation (DO) was determined by quantitative measurements of aldehyde(using a 3-methyl-2-benothiazolinone hydrazone (MBTH) assay) andpolysaccharide (using an anthrone assay).

Thiolation of the oxidized polysaccharide and conjugation were carriedout by the procedure described in example 18 as described above.

Results

Characterization and process data for representative Pn-33F eTAANglycoconjugates using either 2-(2-aminoethoxy)ethane-1-thiol (AEET) orcystamine as spacer and conjugated to CRM₁₉₇ are provided in Table 18.

TABLE 18 Experimental Parameters and Characterization Data for Pn-33FeTAAN conjugates Conjugate Batch Pn-33F-eTAAN- Pn-33F-eTAAN- NalO₄-NalO₄- Cystamine AEET Polysaccharide Mw 1565 kDa 1565 kDa (SEC-MALLS)Poly Oxidation NalO₄ (mol. eq.) 0.14 0.14 Yield 69% 69% Degree ofOxidation 12 12 Thiolation Linker Cystamine AEET NaCNBH3 (mol. eq.) 1.51.5 Yield 100%  100%  Thiol content (mol of thiol/mol of 0.06 0.06polysaccharide) Conjugation to CRM197 Input ratio (SPR) 0.8 0.8Conjugate Results Saccharide yield (%) 56% 59% Output Ratio (SPR) 1.92.2 Free Saccharide <5% <5% Free Protein <1% <1% Conjugate Mw(SEC-MALLS) 6324 kDa 5890 kDa

Polysaccharide-protein conjugates were generated with each of theselinkers having good quality attributes including molecular weight, highyield, low free saccharide and low free protein levels.

Example 20 Process for Preparation of (((2-Oxoethyl)Thio)Alkyl)Amide(Oxo-eTAAD) Linked Glycoconjugate

Pn-22F polysaccharide is compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture is shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized Pn-22F polysaccharide is reconstituted inanhydrous dimethylsulfoxide (DMSO). To this solution, cystaminehydrochloride (1.0 meq) is added, followed by1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.1 meq)and HOBt (1.1 meq). The mixture is then stirred for 18±2 hours at 23±2°C. before it is diluted 10-fold by pouring into pre-chilled sodiumphosphate buffer, pH 6.0. The diluted solution is filtered through a 5μm filter. Dialfiltration of derivatized Pn-22F polysaccharide iscarried out with 100K MWCO ultrafilter membrane cassettes, against Waterfor Injection (WFI). The retentate is collected.

Reduction and Purification of Activated Thiolated Pn-22F Polysaccharide

To the retentate a solution of tris(2-carboxyethyl)phosphine (TCEP, 5mol. eq.), is added after dilution by 10% volume of 0.1M sodiumphosphate buffer, pH 6.0. This reduction reaction is allowed to proceedfor 2±1 hours at 23±2° C. Dialfiltration of thiolated Pn-22Fpolysaccharide is carried out with 100K MWCO ultrafilter membranecassettes. Diafiltration is performed against pre-chilled 10 mM sodiumphosphate, pH 4.3. The thiolated Pn-22F polysaccharide retentate ispulled for both saccharide concentration and thiol (Ellman) assays.

Conjugation of Thiolated Pn-22F Polysaccharide to Bromoacetylated CRM₁₉₇

The CRM₁₉₇ carrier protein is activated separately by bromoacetylation,as described in Example 1, and then reacted with the activated Pn-22Fpolysaccharide for the conjugation reaction. Before starting theconjugation reaction, the reaction vessel is pre-cooled to 5±3° C.Bromoacetylated CRM₁₉₇ and thiolated Pn-22F polysaccharide are mixedtogether in a reaction vessel at an agitation speed of 150-200 rpm,using a saccharide/protein input ratio of 0.9±0.1. The reaction pH isadjusted to 8.0-9.0. The conjugation reaction is allowed to proceed at5±3° C. for 20±2 hours.

Capping of Reactive Groups on Bromoacetylated CRM₁₉₇ and ThiolatedPn-22F Polysaccharide

The unreacted bromoacetylated residues on CRM₁₉₇ proteins are capped byreacting with 2 mol. eq. of N-acetyl-L-cysteine for 3 hours at 5±3° C.,followed by capping any residual free sulfhydryl groups of the thiolatedPn8-polysaccharide with 4 mol. eq. of iodoacetamide (IAA) for 20 hoursat 5±3° C.

Purification of eTAAD-Linked Pn-22F Glycoconjugate

The conjugation solution is filtered through a 0.45 μm or 5 μm filter.Dialfiltration of the Pn-22F glycoconjugate is carried out with 100KMWCO ultrafilter membrane cassettes. Diafiltration is performed against5 mM succinate-0.9% saline, pH 6.0. The Pn-22F glycoconjugate 100Kretentate is then filtered through a 0.22 μm filter and stored at 5±3°C.

Example 21 Process for Preparation of (((2-Oxoethyl)Thio)Alkyl)Amide(Oxo-eTAAD) Linked Glycoconjugate Via Thiazolidinone Activation RouteDerivatization of Pn-22F Polysaccharide

Pn-22F polysaccharide is compounded with 500 mM of 1,2,4-triazole (inWFI) to obtain 10 grams of triazole per gram of polysaccharide. Themixture is shell-frozen in dry ice-ethanol bath and then lyophilized todryness. The lyophilized Pn-22F polysaccharide is reconstituted inanhydrous dimethylsulfoxide (DMSO). To this solution,thiazolidine-2-thione (TT) (0.3 MEq) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.3 meq)solutions in DMSO are added. The mixture is then stirred for 18±2 hoursat 23±2° C. To this solution 0.2 MEq cystamine hydrochloride is addedand the reaction is continued for another 18 hours. The thiolatedpolysaccharide is buffer exchanged with water.

To the retentate a solution of tris(2-carboxyethyl)phosphine (TCEP, 5mol. eq.), is added after dilution by 10% volume of 0.1M sodiumphosphate buffer, pH 6.0. This reduction reaction is allowed to proceedfor 2±1 hours at 23±2° C. Dialfiltration of thiolated Pn8 polysaccharideis carried out with 100K MWCO ultrafilter membrane cassettes.Diafiltration is performed against pre-chilled 10 mM sodium phosphate,pH 4.3. The thiolated Pn-22F polysaccharide retentate is pulled for bothsaccharide concentration and thiol (Ellman) assays.

Conjugation of Thiolated Pn-22F Polysaccharide to Bromoacetylated CRM₁₉₇

Conjugation of the Pn-22F thiolated polysaccharide is carried out asoutlined in Example 20 above.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are herebyincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

1. A glycoconjugate comprising a saccharide covalently conjugated to acarrier protein through a spacer containing ((2-oxoethyl)thio)).
 2. Theglycoconjugate of claim 1 having the general formula (I):

wherein: A is a group (C═X)m wherein X is S or O and m is 0 or 1; B is abond, O, or CH₂; and when m is 0, B can also be (C═O); R is a C₂-C₁₆alkylene, C₂-C₁₆ heteroakylene, NH—C(═O)—C₂-C₁₆ alkylene, orNH—C(═O)—C₂-C₁₆ heteroakylene, wherein said alkylene and heteroalkyleneare optionally substituted by 1, 2 or 3 groups independently selectedfrom COOR′ where R′ is selected from H, methyl, ethyl or propyl; withthe proviso that said spacer is not the(2-((2-oxoethyl)thio)ethyl)carbamate (eTec) spacer. 3.-9. (canceled) 10.The glycoconjugate of claim 2 wherein R is selected from the groupconsisting of (CH₂)₂, (CH₂)₃, (CH₂)₄, (CH₂)₅, (CH₂)₆, (CH₂)₇, (CH₂)₈,(CH₂)₉ and (CH₂)₁₀.
 11. (canceled)
 12. The glycoconjugate of claim 2wherein R is selected from the group consisting of O—CH₂, O—CH₂—CH₂,CH₂—CH₂—O—CH₂—CH₂, O—CH₂—CH₂—O—CH₂—CH₂, O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂,O—CH₂—CH₂—(N—CH₃)—CH₂—CH₂—O—CH₂—CH₂, CH₂—CH₂—(N—CH₃)—CH₂—CH₂ andCH₂—CH₂—S—CH₂—CH₂.
 13. (canceled)
 14. The glycoconjugate of claim 1having the general formula (II):

wherein R is (CH₂)_(n), where n is 3 to
 10. 15.-18. (canceled)
 19. Theglycoconjugate of claim 1 having the general formula (III):

wherein R is selected from (CH₂CH₂O)_(m)CH₂CH₂, CH(COOH)(CH₂)_(n),NHCO(CH₂)_(n), NHCO(CH₂CH₂O)_(m)CH₂CH₂, OCH₂(CH₂)_(n) orO(CH₂CH₂O)_(m)CH₂CH₂; wherein n is selected from 1 to 10 and m isselected from 1 to
 4. 20.-32. (canceled)
 33. The glycoconjugate of claim1 having the general formula (IV):

wherein R is selected from CH₂(CH₂)_(n), (CH₂CH₂O)_(m)CH₂CH₂,CH(COOH)(CH₂)_(n), NHCO(CH₂)_(n), NHCO(CH₂CH₂O)_(m)CH₂CH₂, OCH₂(CH₂)_(n)or O(CH₂CH₂O)_(m)CH₂CH₂; wherein n is selected from 1 to 10 and m isselected from 1 to
 4. 34.-47. (canceled)
 48. The glycoconjugate of claim1 having the general formula (V):

wherein R is selected from CH₂(CH₂)_(n), (CH₂CH₂O)_(m)CH₂CH₂,CH(COOH)(CH₂)_(n), NHCO(CH₂)_(n), NHCO(CH₂CH₂O)_(m)CH₂CH₂, OCH₂(CH₂)_(n)or O(CH₂CH₂O)_(m)CH₂CH₂; wherein n is selected from 1 to 10 and m isselected from 1 to
 4. 49.-62. (canceled)
 63. The glycoconjugate of claim1 comprising a saccharide conjugated to a carrier protein through anoxo-eTAC spacer, wherein the saccharide is covalently linked to theoxo-eTAC spacer through a carbamate linkage, and wherein the carrierprotein is covalently linked to the oxo-eTAC spacer through a thioetherand amide linkage.
 64. The glycoconjugate of claim 1 comprising asaccharide conjugated to a carrier protein through an oxo-eTAAN spacer,wherein the saccharide is covalently linked to the oxo-eTAAN spacerthrough an amine linkage, and wherein the carrier protein is covalentlylinked to the oxo-eTAAN spacer through a thioether and amide linkage.65. The glycoconjugate of claim 1 comprising a saccharide conjugated toa carrier protein through an oxo-eTAAD spacer, wherein the saccharide iscovalently linked to the oxo-eTAAD spacer through an amide linkage, andwherein the carrier protein is covalently linked to the oxo-eTAAD spacerthrough a thioether and amide linkage.
 66. (canceled)
 67. Theglycoconjugate of claim 1, wherein the saccharide is a capsularpolysaccharide derived from bacteria.
 68. The glycoconjugate of claim67, wherein said capsular polysaccharide is derived from S. pneumoniae.69. The glycoconjugate of claim 68, wherein said capsular polysaccharideis selected from the group consisting of Pn-serotype 1, 2, 3, 4, 5, 6A,6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20,22F, 23A, 23B, 23F, 33F and 35B capsular polysaccharides. 70.-87.(canceled)
 88. The glycoconjugate of claim 1, wherein the saccharide hasa molecular weight of between 10 kDa and 2,000 kDa.
 89. (canceled) 90.The glycoconjugate of claim 1, wherein the glycoconjugate has amolecular weight of between 50 kDa and 20,000 kDa.
 91. (canceled) 92.The glycoconjugate of claim 67, wherein the polysaccharide has a degreeof O-acetylation between 75-100%.
 93. The glycoconjugate of claim 1,wherein the carrier protein is selected from the group consisting of TT,DT, DT mutants, H. influenzae protein D, PhtX, PhtD, PhtE fusions,detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B ofC. difficile, and PsaA. 94.-104. (canceled)
 105. The glycoconjugate ofclaim 1, wherein the carrier protein is CRM₁₉₇. 106.-119. (canceled)120. A method of making a glycoconjugate comprising a saccharideconjugated to a carrier protein through a(((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC) spacer, comprising thesteps of: a) reacting a saccharide with a carbonic acid derivative orcyanogen derivative, to produce an activated saccharide; b) reacting theactivated saccharide with a bifunctional linker containing amine andthiol functionalities or a salt thereof, to produce a thiolatedsaccharide; c) reacting the thiolated saccharide with a deprotecting orreducing agent to produce an activated thiolated saccharide comprisingone or more free sulfhydryl residues; d) reacting the activatedthiolated saccharide with an activated carrier protein comprising one ormore α-haloacetamide groups, to produce a thiolated saccharide-carrierprotein conjugate; and e) reacting the thiolated saccharide-carrierprotein conjugate with (i) a first capping reagent capable of cappingunconjugated α-haloacetamide groups of the activated carrier protein;and/or (ii) a second capping reagent capable of capping unconjugatedfree sulfhydryl residues of the activated thiolated saccharide;producing an oxo-eTAC linked glycoconjugate. 121.-128. (canceled) 129.The method of claim 120, wherein step e) comprises capping withN-acetyl-L-cysteine as the first capping reagent and IAA as the secondcapping reagent. 130.-133. (canceled)
 134. A method of making aglycoconjugate comprising a saccharide conjugated to a carrier proteinthrough an (((2-oxoethyl)thio)alkyl)amine (oxo-eTAAN) spacer, comprisingthe steps of: a) reacting a saccharide with an oxidizing reagent togenerate aldehyde groups to produce an activated saccharide; b) reactingthe activated saccharide with a bifunctional linker containing amine andthiol functionalities (in protected or free forms) from the amino end ofthe linker, to produce a thiolated saccharide by reductive amination; c)reacting the thiolated saccharide with a deprotecting agent or reducingagent (if thiol is protected) to produce an activated thiolatedsaccharide comprising one or more free sulfhydryl residues; d) reactingthe activated thiolated saccharide with an activated carrier proteincomprising one or more α-haloacetamide groups, to produce a thiolatedsaccharide-carrier protein conjugate; and e) reacting the thiolatedsaccharide-carrier protein conjugate with (i) a first capping reagentcapable of capping unconjugated α-haloacetamide groups of the activatedcarrier protein; and/or (ii) a second capping reagent capable of cappingunconjugated free sulfhydryl residues of the activated thiolatedsaccharide; producing an oxo-eTAAN linked glycoconjugate. 135.(canceled)
 136. A method of making a comprising a carboxyl containingsaccharide conjugated to a carrier protein through an(((2-oxoethyl)thio)alkyl)amide (oxo-eTAAD) spacer, comprising the stepsof: a) reacting the carboxyl group containing saccharide to generated anactivated saccharide with a carbodiimide or a derivative thereof; b)reacting the activated saccharide with a heterobifunctional linkercontaining amine and thiol functionalities (in protected or free form)from the amino end, to produce a thiolated saccharide; c) reacting thethiolated saccharide with a deprotecting or reducing agent (ifprotected) to produce an activated thiolated saccharide comprising oneor more free sulfhydryl residues; d) reacting the activated thiolatedsaccharide with an activated carrier protein comprising one or moreα-haloacetamide groups, to produce a thiolated saccharide-carrierprotein conjugate; and e) reacting the thiolated saccharide-carrierprotein conjugate with (i) a first capping reagent capable of cappingunconjugated α-haloacetamide groups of the activated carrier protein;and/or (ii) a second capping reagent capable of capping unconjugatedfree sulfhydryl residues of the activated thiolated saccharide;producing an oxo-eTAAD linked glycoconjugate. 137.-167. (canceled)